Oligonucleotide compounds comprising non-nucleotide overhangs

ABSTRACT

The invention relates to siRNA compounds comprising one non-nucleotide moiety covalently attached to at least one of the sense or antisense strands to down-regulate the expression of human genes. The invention also relates to pharmaceutical compositions comprising such compounds and a pharmaceutically acceptable carrier and to methods of treating and/or preventing the incidence or severity of various diseases or conditions associated with the target genes and/or symptoms associated with such diseases or conditions.

Throughout this application various patent and scientific publicationsare cited. The disclosures for these publications in their entiretiesare hereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

CROSS REFERENCED TO RELATED APPLICATIONS

This is a Continuation of co-pending U.S. patent application Ser. No.13/521,033 filed Jul. 6, 2012, which is the National Stage of PCT PatentApplication No. PCT/US2011/020298 filed Jan. 6 2011, which claimedpriority to PCT Patent Application No. PCT/US2010/059578 filed Dec. 8,2010, PCT Patent Application No. PCT/US2010/049047 filed Sep. 16, 2010,and U.S. Provisional Patent Application No. 61/292,878 filed Jan. 7,2010; all of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

Disclosed herein are modified double stranded nucleic acid molecules,pharmaceutical compositions comprising same and methods of use thereoffor the inhibition of mammalian and non-mammalian target genes. Thecompounds and compositions are thus useful in the treatment of subjectssuffering from diseases or conditions and or symptoms associated withsuch diseases or conditions in which gene expression has adverseconsequences. In particular embodiments the invention providescompositions comprising same and methods of use thereof.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) in mammals is mediated by small interfering RNAs(siRNAs) (Fire et al, Nature 1998, 391:806) or microRNAs (miRNAs)(Ambros, Nature 2004, 431(7006):350-355; Bartel, Cell 2004, 116(2):281-97). The corresponding process in plants is commonly referred to asspecific post-transcriptional gene silencing (PTGS) or RNA silencing andis also referred to as quelling in fungi.

A siRNA is a double-stranded RNA or modified RNA molecule whichdown-regulates or silences (prevents) the expression of a gene/mRNA ofits endogenous (cellular) counterpart. The mechanism of RNA interferenceis detailed infra.PCT Publication No. WO 2008/050329 and U.S. Ser. No. 11/978,089 to theassignee of the present invention relate to inhibitors of pro-apoptoticgenes, and are incorporated by reference in their entirety. PCT PatentPublication Nos. WO 2008/104978 and WO 2009/044392 to the assignee ofthe present invention relate to chemically modified siRNA structures,and are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention provides chemically and or structurally modified siRNAcompounds for the inhibition of gene expression in general and ofmammalian and prokaryotic genes, in particular. Provided herein arenovel structural motifs useful in the preparation of siRNAoligonucleotides comprising one or more non-nucleotide moieties as a 3′(or 2′) overhang at the 3′-terminus, compositions comprising same andmethods of use thereof. The applicant has determined that the additionof one, and preferably two or three, non-nucleotide moieties to the 3′terminus of a siRNA provides advantageous properties to the siRNA interms of activity and or stability and or delivery. Accordingly, anexisting siRNA can be advantageously modified and future siRNA can bedesigned and produced to take advantage of this finding. Without wishingto be bound to theory, the nucleic acid molecules disclosed herein andhaving a 3′ non-nucleotide overhang (Z or Z′ i.e. C3Pi-C3Ps; C3Pi-C3OH;C3Pi-C3Pi; C3Pi-rAb; C3Pi-dAb) are recognized by the PAZ domain ofArgonaute and are able to perform RNAi, while exhibiting good stabilityand activity.

It will be appreciated that although the term “3′ terminus” is usedthroughout this application to refer to the end of the siRNA strand towhich the non-nucleotide moieties are attached, unless explicitly statedotherwise the non-nucleotide moieties, or “overhangs” may be attached atthe 3′ position of the (deoxy)ribose moiety at the 3′-terminus of theoligonucleotide strand or at the 2′ position of the (deoxy)ribose moietyat the 3′-terminus of the oligonucleotide strand, e.g.

(attachment at the 3′ position of the 3′-terminus (deoxy)ribose moiety;in the drawing, B is a nucleotide base and R is H or OH)or

(attachment at the 2′ position of the 3′-terminus (deoxy)ribose moiety).

Provided herein are novel structures of double stranded nucleic acidmolecules, having advantageous properties and which may be applied tosiRNA to any target sequence, comprising non-nucleotide overhangs at oneor both 3′ termini of the duplex. The chemically modified siRNAmodifications disclosed herein are useful in the preparation of stableand active siRNA compounds useful in RNA interference (RNAi).

The application also provides pharmaceutical compositions comprising oneor more such oligonucleotides and methods for treating or preventing theincidence or severity of a disease or condition in a subject in needthereof wherein the disease or condition and/or symptoms associatedtherewith is associated with expression of the target gene. In someembodiments the disease or condition is selected from the groupconsisting of hearing loss, acute renal failure (ARF), glaucoma, acuterespiratory distress syndrome (ARDS) and other acute lung andrespiratory injuries, ischemia-reperfusion injury following lungtransplantation, ocular ischemic conditions, organ transplantationincluding lung, liver, heart, pancreas, and kidney transplantationincluding delayed graft function (DGF), nephro- and neurotoxicity,spinal cord injury, pressure sores, age-related macular degeneration(AMD), dry eye syndrome, oral mucositis, ischemic ocular neuropathy(ION) and chronic obstructive pulmonary disease (COPD). Such methodsinvolve administering to a mammal in need of such treatment aprophylactically or therapeutically effective amount of one or more suchcompounds, which inhibit or reduce expression or activity of at leastone such gene. Such compounds can be administered concurrently or inplace of other treatments.

The oligonucleotide is selected to target any mammalian or non-mammaliangene. In various embodiments the modified compound comprises anoligonucleotide sequence set forth in any one of SEQ ID NOS:97-68654(disclosed in U.S. Ser. No. 11/978,089 and PCT Patent Application No.PCT/IL 2007/001278, which are hereby incorporated by reference in theirentirety).

In one aspect a double stranded siRNA compound comprising at least onenon-nucleotide 3′ terminal overhang is provided. The applicationprovides a synthetic double stranded siRNA compound comprising a sensestrand and an antisense strand, wherein at least one of the sense orantisense strands comprises 1, 2, 3, 4, or 5 non-nucleotide moieties,preferably 2 or 3, covalently attached at the 3′ terminal end whereinthe non-nucleotide moiety is selected from an inverted abasic moiety, anabasic moiety, an alkyl (hydrocarbon) moiety or derivatives thereof, anda phosphate based moiety. In some embodiments the non-nucleotide moietyis selected from an inverted abasic moiety, an alkyl (hydrocarbon)moiety or derivatives thereof and a phosphate based moiety. In someembodiments the non-nucleotide moiety comprises an alkyl (hydrocarbon)moiety or a derivative thereof. Provided herein is a double strandednucleic acid molecule which includes a sense strand and an antisensestrand, wherein at least one strand comprises a non-nucleotide moietycovalently attached at a 3′ or a 2′ position of the sugar residue at the3′ terminal nucleotide of the strand in which it is present; wherein thenon-nucleotide moiety is selected from the group consisting of propanol,a C3 alkyl moiety linked to a phosphodiester, a C3 alkyl moiety linkedto a phosphorothioate, a deoxyriboabasic moiety a riboabasic moiety anda combination thereof.

In some embodiments the non-nucleotide moiety is attached to the sugarresidue via a phosphate base linkage, preferably a phosphodiester or aphosphorothioate linkage. In some embodiments the non-nucleotide moietyincludes a C3 alkyl moiety covalently attached at a 3′ or a 2′ positionof the sugar residue at the 3′ terminus of the antisense strand. Invarious embodiments the C3 alkyl moiety is selected from C3Pi and C3OH.

In some embodiments the molecule includes two or three C3 alkyl moietiescovalently linked by a phosphodiester or phosphorothioate linkage or oneC3 alkyl moiety covalently linked by a phosphodiester orphosphorothioate linkage to an abasic moiety. In preferred embodimentsthe molecule includes two C3 alkyl moieties covalently linked by aphosphodiester or phosphorothioate linkage.

In some embodiments the C3 alkyl moieties are selected from C3Pi-C3OH,C3Pi-C3Pi, C3Pi-C3Ps, C3Pi-C3Pi-C3OH, C3Ps-C3Ps-C3OH, C3Pi-C3Ps-C3OH,C3Ps-C3Pi-C3OH, C3Pi-C3Pi-C3Pi, C3Ps-C3Ps-C3Ps, C3Pi-C3Ps-C3Ps,C3Ps-C3Pi-C3Ps, C3Ps-C3Ps-C3Pi, C3Pi-C3Pi-C3Ps, C3Ps-C3Pi-C3Pi orC3Pi-C3Ps-C3Pi moieties.

In other embodiments the molecule includes a C3 alkyl moiety covalentlylinked by a phosphodiester or phosphorothioate linkage to an abasicmoiety wherein the abasic moiety is selected from a deoxyriboabasicmoiety or a riboabasic moiety. In some embodiments the C3 alkyl moietycovalently linked by a phosphodiester or phosphorothioate linkage to anabasic moiety is selected from C3Pi-rAb, C3Pi-dAb, rAb-C3OH, rAb-C3Pi,dAb-C3OH, or dAb-C3Pi.

In some embodiments, provided are double stranded nucleic acid moleculeshaving the structure (A1):

(A1) 5′ (N)x-Z3′     (antisense strand) 3′ Z′-(N′)y-z″5′(sense strand)wherein each of N and N′ is a nucleotide which may be unmodified ormodified, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein at least one of Z or Z′ is present and comprises anon-nucleotide moiety covalently attached at the 3′ terminus of thestrand in which it is present;wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;wherein each of x and y is independently an integer between 18 and 40;wherein the sequence of (N′)y has complementarity to the sequence of(N)x; and wherein the sequence of (N)x has complementarity to aconsecutive sequence in a target RNA.

In some embodiments the covalent bond joining each consecutive N or N′is a phosphodiester bond.

In some embodiments x=y=19 to 27, for example 19, 20, 21, 22, 23, 24,25, 26, 27. In some embodiments x=y and each of x and y is 19, 20, 21,22 or 23. In various embodiments x=y=19.

In some embodiments x=y=19 and one of Z or Z′ is present and consists oftwo non-nucleotide moieties.

In some embodiments x=y=19 and Z′ is present and consists of twonon-nucleotide moieties.

In preferred embodiments x=y=19 and Z is present and consists twonon-nucleotide moieties.

In preferred embodiments x=y=19 and Z is present and consists of twonon-nucleotide moieties; and Z′ is present and consists of onenon-nucleotide moiety.

In additional embodiments x=y=19 and Z and Z′ are present and eachindependently comprises two non-nucleotide moieties.

In some embodiments the double stranded nucleic acid molecules comprisea DNA moiety or a mismatch to the target at position 1 of the antisensestrand (5′ terminus). Such a structure is described herein. According toone embodiment provided are double stranded nucleic acid moleculeshaving a structure (A2) set forth below:

(A2) 5′ N1-(N)x-Z3′     (antisense strand) 3′Z′-N2-(N′)y-z″5′(sense strand)wherein each of N2, N and N′ is an unmodified or modifiedribonucleotide, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;wherein each of x and y is independently an integer between 17 and 39;wherein the sequence of (N′)y has complementarity to the sequence of(N)x and (N)x has complementarity to a consecutive sequence in a targetRNA;wherein N1 is covalently bound to (N)x and is mismatched to the targetRNA or is a complementary DNA moiety to the target RNA;wherein N1 is a moiety selected from the group consisting of natural ormodified uridine, deoxyribouridine, ribothymidine, deoxyribothymidine,adenosine or deoxyadenosine;wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N2-(N′)y; andwherein at least one of Z or Z′ is present and comprises anon-nucleotide moiety covalently attached at the 3′ terminus of thestrand in which it is present.

In some embodiments x=y=18 and one of Z or Z′ is present and consists oftwo non-nucleotide moieties.

In some embodiments x=y=18 and Z′ is present and consists of twonon-nucleotide moieties.

In preferred embodiments x=y=18 and Z is present and consists twonon-nucleotide moieties.

In preferred embodiments x=y=18 and Z is present and consists of twonon-nucleotide moieties; and Z′ is present and consists of onenon-nucleotide moiety.

In additional embodiments x=y=18 and Z and Z′ are present and eachindependently comprises two non-nucleotide moieties.

In some embodiments the sequence of (N′)y is fully complementary to thesequence of (N)x. In various embodiments sequence of N2-(N′)y iscomplementary to the sequence of N1-(N)x. In some embodiments (N)xcomprises an antisense that is fully complementary to about 17 to about39 consecutive nucleotides in a target RNA. In other embodiments (N)xcomprises an antisense that is substantially complementary to about 17to about 39 consecutive nucleotides in a target RNA.

In some embodiments N1 and N2 form a Watson-Crick base pair. In someembodiments N1 and N2 form a non-Watson-Crick base pair. In someembodiments a base pair is formed between a ribonucleotide and adeoxyribonucleotide.

In some embodiments x=y=18, x=y=19 or x=y=20. In preferred embodimentsx=y=18. When x=18 in N1-(N)x, N1 refers to. position 1 and positions2-19 are included in (N)18 When y=18 in N2-(N′)y N2. refers to position19 and positions 1-18 are included in (N′)18

In some embodiments N1 is covalently bound to (N)x and is mismatched tothe target RNA. In various embodiments N1 is covalently bound to (N)xand is a DNA moiety complementary to the target RNA.

In some embodiments N1 and N2 form a base pair between uridine ordeoxyuridine and adenosine or deoxyadenosine (rU-rA, rU-dA, dU-rA,dU-dA). In other embodiments N1 and N2 form a base pair betweendeoxyuridine and adenosine.

In some embodiments the double stranded nucleic acid molecule is asiRNA, siNA or a miRNA. The double stranded nucleic acid molecules asprovided herein are also referred to as “duplexes”.

In certain preferred embodiments x=y=18. In some embodiments N1 and N2form a Watson-Crick base pair. In other embodiments N1 and N2 form anon-Watson-Crick base pair. In certain embodiments N1 is selected fromthe group consisting of riboadenosine, modified riboadenosine,deoxyriboadenosine, modified deoxyriboadenosine. In other embodiments N1is selected from the group consisting of ribouridine, deoxyribouridine,modified ribouridine, and modified deoxyribouridine.

In some embodiments each of N and N′ is an unmodified nucleotide. Insome embodiments at least one of N or N′ includes a chemically modifiednucleotide or an unconventional moiety. In some embodiments theunconventional moiety is selected from a mirror nucleotide, an abasicribose moiety and an abasic deoxyribose moiety. In some embodiments theunconventional moiety is a mirror nucleotide, preferably an L-DNAmoiety. In some embodiments at least one of N or N′ includes a 2′OMesugar-modified ribonucleotide.

In some embodiments the sequence of (N′)y is fully complementary to thesequence of (N)x. In other embodiments the sequence of (N′)y issubstantially complementary to the sequence of (N)x.

In some embodiments (N)x includes an antisense sequence that is fullycomplementary to about 17 to about 39 consecutive nucleotides in atarget RNA. In other embodiments (N)x includes an antisense that issubstantially complementary to about 17 to about 39 consecutivenucleotides in a target RNA.

In some embodiments the nucleic acid molecules disclosed herein aresiRNA, siNA or miRNA.

In some embodiments of Structures A1 and A2, Z is present and Z′ isabsent. In other embodiments Z′ is present and Z is absent. Inadditional embodiments both Z and Z′ are present. In some embodiments Zand Z′ are present and are identical. In further embodiments Z and Z′are present and are different. In some embodiments Z and Z′ areindependently 2, 3, 4 or 5 non-nucleotide moieties or a combination of2, 3, 4, or 5 non-nucleotide moieties and nucleotides. In someembodiments each of Z and or Z′ consist of two (2) non-nucleotidemoieties covalently attached to the 3′ terminus of the siRNA strand viaa phosphodiester bond.

A non-nucleotide moiety is selected from the group consisting of anabasic moiety, an inverted abasic moiety, an alkyl moiety or derivativethereof, and an inorganic phosphate. In some embodiments anon-nucleotide moiety is an alkyl moiety or derivative thereof. In someembodiments the alkyl moiety comprises a terminal functional groupselected from the group consisting of an alcohol, a terminal amine, aterminal phosphate and a terminal phosphorothioate moiety.

In some embodiments Z is present and comprises one or morenon-nucleotide moieties selected from the group consisting of an abasicmoiety, an inverted abasic moiety, hydrocarbon moiety or derivativethereof, and an inorganic phosphate. In some embodiments Z is presentand consists of two alkyl moieties or derivatives thereof.

In additional embodiments Z′ is present and comprises one or morenon-nucleotide moieties selected from the group consisting of an abasicmoiety, an inverted abasic moiety, a hydrocarbon moiety, and aninorganic phosphate. In some embodiments Z′ is present and comprises oneor more alkyl moieties or derivatives thereof.

In some embodiments Z is present and consists of two alkyl moieties orderivatives thereof and Z′ is present and consists of a single alkylmoiety or derivative thereof.

In some embodiments each of Z and Z′ includes an abasic moiety, forexample a deoxyriboabasic moiety (referred to herein as “dAb”) orriboabasic moiety (referred to herein as “rAb”). In some embodimentseach of Z and/or Z′ comprises two covalently linked abasic moieties andis for example 5′>3′ dAb-dAb or rAb-rAb or dAb-rAb or rAb-dAb. Eachmoiety is covalently conjugated to an adjacent moiety via a covalentbond, preferably a phospho-based bond. In some embodiments thephospho-based bond is a phosphorothioate, a phosphonoacetate or aphosphodiester bond.

In some embodiments each of Z and/or Z′ independently includes a C2, C3,C4, C5 or C6 alkyl moiety, optionally a C3 [propane, —(CH2)₃-] moiety ora derivative thereof e.g. propanol (C3-OH), propanediol, orphosphodiester derivative of propanediol (“C3Pi”). In preferredembodiments each of Z and/or Z′ includes two hydrocarbon moieties and insome examples is C3-C3. Each C3 is covalently conjugated to an adjacentC3 via a covalent bond, preferably a phospho-based bond. In someembodiments the phospho-based bond is a phosphorothioate, aphosphonoacetate or a phosphodiester bond.

In some embodiments of Structure A1 and Structure A2 at least one of Zor Z′ is present and comprises at least two non-nucleotide moietiescovalently attached to the strand in which it is present. In someembodiments each of Z and Z′ independently includes a C3 alkyl, C3alcohol or C3 ester moiety. In some embodiments Z′ is absent and Z ispresent and includes a non-nucleotide C3 moiety. In some embodiments Zis absent and Z′ is present and includes a non-nucleotide C3 moiety.

In some embodiments of Structures A1 and A2, each of N and N′ is anunmodified nucleotide. In some embodiments at least one of N or N′includes a chemically modified nucleotide or an unconventional moiety.In some embodiments the unconventional moiety is selected from a mirrornucleotide, an abasic ribose moiety and an abasic deoxyribose moiety. Insome embodiments the unconventional moiety is a mirror nucleotide,preferably an L-DNA moiety. In some embodiments at least one of N or N′includes a 2′OMe sugar-modified ribonucleotide.

In some embodiments the sequence of (N′)y is fully complementary to thesequence of (N)x. In other embodiments the sequence of (N′)y issubstantially complementary to the sequence of (N)x.

In other embodiments the compound of Structure A1 or Structure A2includes at least one ribonucleotide modified in the sugar residue. Insome embodiments the compound includes a modification at the 2′ positionof the sugar residue. In some embodiments the modification in the 2′position includes the presence of an amino, a fluoro, an alkoxy or analkyl moiety. In certain embodiments the 2′ modification includes analkoxy moiety, In preferred embodiments the alkoxy moiety is a methoxymoiety (also known as 2′-O-methyl; 2′OMe; 2′-OCH3). In some embodimentsthe nucleic acid compound includes 2′OMe sugar modified alternatingribonucleotides in one or both of the antisense and the sense strands.In other embodiments the compound includes 2′OMe sugar modifiedribonucleotides in the antisense strand, (N)x or N1-(N)x, only. Incertain embodiments the middle ribonucleotide of the antisense strand;e.g. ribonucleotide in position 10 in a 19-mer strand is unmodified. Invarious embodiments the nucleic acid compound includes at least 5alternating 2′OMe sugar modified and unmodified ribonucleotides. Inadditional embodiments the compound of Structure A1 or Structure A2includes modified ribonucleotides in alternating positions wherein eachribonucleotide at the 5′ and 3′ termini of (N)x or N1-(N)x are modifiedin their sugar residues, and each ribonucleotide at the 5′ and 3′termini of (N′)y or N2-(N)y are unmodified in their sugar residues.

In some embodiments the double stranded molecule includes one or more ofthe following modifications

a) N in at least one of positions 5, 6, 7, 8, or 9 from the 5′ terminusof the antisense strand is selected from a 2′5′ nucleotide or a mirrornucleotide;b) N′ in at least one of positions 9 or 10 from the 5′ terminus of thesense strand is selected from a 2′5′ nucleotide and a pseudoUridine; andc) N′ in 4, 5, or 6 consecutive positions at the 3′ terminus positionsof (N′)y comprises a 2′5′ nucleotide.

In some embodiments the double stranded molecule includes a combinationof the following modifications

a) the antisense strand includes a 2′5′ nucleotide or a mirrornucleotide in at least one of positions 5, 6, 7, 8, or 9 from the 5′terminus; andb) the sense strand includes at least one of a 2′5′ nucleotide and apseudoUridine in positions 9 or 10 from the 5′ terminus.

In some embodiments the double stranded molecule includes a combinationof the following modifications

a) the antisense strand includes a 2′5′ nucleotide or a mirrornucleotide in at least one of positions 5, 6, 7, 8, or 9 from the 5′terminus; andc) the sense strand includes 4, 5, or 6 consecutive 2′5′ nucleotides atthe 3′ penultimate or 3′ terminal positions.

In some embodiments, the sense strand [(N)x or N1-(N)x] includes 1, 2,3, 4, 5, 6, 7, 8, or 9 2′OMe sugar modified ribonucleotides. In someembodiments, the antisense strand includes 2′OMe modifiedribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17 and 19. In otherembodiments antisense strand includes 2′OMe modified ribonucleotides atpositions 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. In other embodiments theantisense strand includes 2′OMe modified ribonucleotides at positions 3,5, 7, 9, 11, 13, 15, 17 and 19. In some embodiments the antisense strandincludes one or more 2′OMe sugar modified pyrimidines. In someembodiments all the pyrimidine nucleotides in the antisense strand are2′OMe sugar modified. In some embodiments the sense strand includes2′OMe sugar modified pyrimidines.

In some embodiments of Structure A1 and Structure A2, the sense strandand the antisense strand are independently phosphorylated orunphosphorylated at the 3′ terminus and at the 5′ terminus. In someembodiments of Structure A1 and Structure A2, the sense strand and theantisense strand are unphosphorylated at the 3′ and 5′ termini. In otherembodiments the sense strand and the antisense strand are phosphorylatedat the 3′ termini.

In some embodiments of Structure A1 and Structure A2 (N)y includes atleast one unconventional moiety selected from a mirror nucleotide, a2′5′ nucleotide and a TNA. In some embodiments the unconventional moietyis a mirror nucleotide. In various embodiments the mirror nucleotide isselected from an L-ribonucleotide (L-RNA) and an L-deoxyribonucleotide(L-DNA). In preferred embodiments the mirror nucleotide is L-DNA. Incertain embodiments the sense strand comprises an unconventional moietyin position 9 or 10 (from the 5′ terminus). In preferred embodiments thesense strand includes an unconventional moiety in position 9 (from the5′ terminus). In some embodiments the sense strand is 19 nucleotides inlength and comprises 4, 5, or 6 consecutive unconventional moieties inpositions 15, (from the 5′ terminus). In some embodiments the sensestrand includes 4 consecutive 2′5′ ribonucleotides in positions 15, 16,17, and 18. In some embodiments the sense strand includes 5 consecutive2′5′ ribonucleotides in positions 15, 16, 17, 18 and 19. In variousembodiments the sense strand further comprises Z′. In some embodimentsZ′ includes a C3OH moiety or a C3Pi moiety.

In some embodiments of Structure A1 (N′)y includes at least one L-DNAmoiety. In some embodiments x=y=19 and (N′)y, consists of unmodifiedribonucleotides at positions 1-17 and 19 and one L-DNA at the 3′penultimate position (position 18). In other embodiments x=y=19 and(N′)y consists of unmodified ribonucleotides at positions 1-16 and 19and two consecutive L-DNA at the 3′ penultimate position (positions 17and 18). In various embodiments the unconventional moiety is anucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotidephosphate linkage. According to various embodiments (N′)y includes 2, 3,4, 5, or 6 consecutive ribonucleotides at the 3′ terminus linked by2′-5′ internucleotide linkages. In one embodiment, four consecutivenucleotides at the 3′ terminus of (N′)y are joined by three 2′-5′phosphodiester bonds, wherein one or more of the 2′-5′ nucleotides whichform the 2′-5′ phosphodiester bonds further includes a 3′-O-methyl(3′OMe) sugar modification. Preferably the 3′ terminal nucleotide of(N′)y includes a 2′OMe sugar modification. In certain embodiments x=y=19and (N′)y includes two or more consecutive nucleotides at positions 15,16, 17, 18 and 19 include a nucleotide joined to an adjacent nucleotideby a 2′-5′ internucleotide bond (2′-5′ nucleotide). In variousembodiments the nucleotide forming the 2′-5′ internucleotide bondincludes a 3′ deoxyribose nucleotide or a 3′ methoxy nucleotide (3′ H or3′OMe in place of a 3′ OH). In some embodiments x=y=19 and (N′)yincludes 2′-5′ nucleotides at positions 15, 16 and 17 such that adjacentnucleotides are linked by a 2′-5′ internucleotide bond between positions15-16, 16-17 and 17-18; or at positions, 15, 16, 17, 18, and 19 suchthat adjacent nucleotides are linked by a 2′-5′ internucleotide bondbetween positions 15-16, 16-17, 17-18 and 18-19 and a 3′OH is availableat the 3′ terminal nucleotide or at positions 16, 17 and 18 such thatadjacent nucleotides are linked by a 2′-5′ internucleotide bond betweenpositions 16-17, 17-18 and 18-19. In some embodiments x=y=19 and (N′)yincludes 2′-5′ nucleotides at positions 16 and 17 or at positions 17 and18 or at positions 15 and 17 such that adjacent nucleotides are linkedby a 2′-5′ internucleotide bond between positions 16-17 and 17-18 orbetween positions 17-18 and 18-19 or between positions 15-16 and 17-18,respectively. In other embodiments the pyrimidine ribonucleotides (rU,rC) in (N′)y are substituted with nucleotides joined to the adjacentnucleotide by a 2′-5′ internucleotide bond. In some embodiments x=y=19and (N′)y comprises five consecutive nucleotides at the 3′ terminusjoined by four 2′-5′ linkages, specifically the linkages between thenucleotides position 15-16, 16-17, 17-18 and 18-19.

In some embodiments x=y=19 and (N′)y comprises five consecutivenucleotides at the 3′ terminus joined by four 2′-5′ linkages andoptionally further includes Z′ and z′ independently selected from aninverted abasic moiety and a C3 alkyl [C3; 1,3-propanediolmono(dihydrogen phosphate)] cap. The C3 alkyl cap is covalently linkedto the 3′ or 5′ terminal nucleotide. In some embodiments the 3′ C3terminal cap further comprises a 3′ phosphate. In some embodiments the3′ C3 terminal cap further comprises a 3′ terminal hydroxyl group.

In some embodiments x=y=19 and (N′)y comprises an L-DNA position 18; and(N′)y optionally further includes Z′ and z′ independently selected froman inverted abasic moiety and a C3 alkyl [C3; 1,3-propanediolmono(dihydrogen phosphate)] cap.

In some embodiments (N′)y comprises a 3′ terminal phosphate (i.e.phosphorylated at the 3′ terminus). In some embodiments (N′)y comprisesa 3′ terminal hydroxyl.

In some embodiments x=y=19 and (N)x includes 2′OMe sugar modifiedribonucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or atpositions 2, 4, 6, 8, 11, 13, 15, 17, 19. In some embodiments x=y=19 and(N)x includes 2′OMe sugar modified pyrimidines. In some embodiments allpyrimidines in (N)x include the 2′OMe sugar modification.

In some embodiments of structure A2 x=y=18 and N2 is a riboadenosinemoiety. In some embodiments x=y=18, and N2-(N′)y comprises fiveconsecutive nucleotides at the 3′ terminus joined by four 2′-5′linkages, specifically the linkages between the nucleotides position15-16, 16-17, 17-18 and 18-19. In some embodiments the linkages includephosphodiester bonds. In some embodiments x=y=18 and N2-(N′)y comprisesfive consecutive nucleotides at the 3′ terminus joined by four 2′-5′linkages and optionally further includes Z′ and z′ independentlyselected from an inverted abasic moiety and a C3 alkyl [C3;1,3-propanediol mono(dihydrogen phosphate)] cap. In some embodimentsx=y=18 and N2-(N′)y comprises an L-DNA position 18; and (N′)y optionallyfurther includes Z′ and z′ independently selected from an invertedabasic moiety and a C3 alkyl [C3; 1,3-propanediol mono(dihydrogenphosphate)] cap. In some embodiments N2-(N′)y comprises a 3′ terminalphosphate. In some embodiments N2-(N′)y comprises a 3′ terminalhydroxyl. In some embodiments x=y=18 and N1-(N)x includes 2′OMe sugarmodified ribonucleotides in positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19or in positions 1, 3, 5, 9, 11, 13, 15, 17, 19, or in positions 3, 5, 9,11, 13, 15, 17, or in positions 2, 4, 6, 8, 11, 13, 15, 17, 19. In someembodiments x=y=18 and N1-(N)x includes 2′OMe sugar modifiedribonucleotides at positions 11, 13, 15, 17 and 19 (from 5′ terminus).In some embodiments x=y=18 and N1-(N)x includes 2′OMe sugar modifiedribonucleotides in positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or inpositions 3, 5, 7, 9, 11, 13, 15, 17, 19. In some embodiments x=y=18 andN1-(N)x includes 2′OMe sugar modified ribonucleotides in positions 2, 4,6, 8, 11, 13, 15, 17, 19.

In some embodiments x=y=18 and N1-(N)x includes 2′OMe sugar modifiedpyrimidines. In some embodiments all pyrimidines in (N)x include the2′OMe sugar modification. In some embodiments the antisense strandfurther comprises an L-DNA or a 2′-5′ nucleotide in position 5, 6 or 7(5′>3′). In other embodiments the antisense strand further comprises aribonucleotide, which generates a 2′5′ internucleotide linkage inbetween the ribonucleotides in positions 5-6 or 6-7 (5′>3′).

In additional embodiments N1-(N)x further includes Z wherein Z comprisesa non-nucleotide overhang. In some embodiments the non-nucleotideoverhang is C3-C3 [1,3-propanediol mono(dihydrogen phosphate)]2.

In some embodiments of Structure A2, (N)y includes at least one L-DNAmoiety. In some embodiments x=y=18 and (N′)y consists of unmodifiedribonucleotides at positions 1-16 and 18 and one L-DNA at the 3′penultimate position (position 17). In other embodiments x=y=18 and(N′)y consists of unmodified ribonucleotides at position 1-15 and 18 andtwo consecutive L-DNA at the 3′ penultimate position (positions 16 and17). In various embodiments the unconventional moiety is a nucleotidejoined to an adjacent nucleotide by a 2′-5′ internucleotide phosphatelinkage. According to various embodiments (N′)y includes 2, 3, 4, 5, or6 consecutive ribonucleotides at the 3′ terminus linked by 2′-5′internucleotide linkages. In one embodiment, four consecutivenucleotides at the 3′ terminus of (N′)y are joined by three 2′-5′phosphodiester bonds, wherein one or more of the 2′-5′ nucleotides whichform the 2′-5′ phosphodiester bonds further includes a 3′-O-methyl(3′OMe) sugar modification. Preferably the 3′ terminal nucleotide of(N′)y includes a 2′OMe sugar modification. In certain embodiments x=y=18and in (N′)y two or more consecutive nucleotides at positions 14, 15,16, 17, and 18 include a nucleotide joined to an adjacent nucleotide bya 2′-5′ internucleotide bond. In various embodiments the nucleotideforming the 2′-5′ internucleotide bond includes a 3′ deoxyribosenucleotide or a 3′ methoxy nucleotide. In some embodiments x=y=18 and(N′)y includes nucleotides joined to the adjacent nucleotide by a 2′-5′internucleotide bond between positions 15-16, 16-17 and 17-18 or betweenpositions 16-17 and 17-18. In some embodiments x=y=18 and (N′)y includesnucleotides joined to the adjacent nucleotide by a 2′-5′ internucleotidebond between positions 14-15, 15-16, 16-17, and 17-18 or betweenpositions 15-16, 16-17, and 17-18 or between positions 16-17 and 17-18or between positions 17-18 or between positions 15-16 and 17-18. Inother embodiments the pyrimidine ribonucleotides (rU, rC) in (N′)y aresubstituted with nucleotides joined to the adjacent nucleotide by a2′-5′ internucleotide bond.

The C3 alkyl moiety may be covalently linked to the 3′ terminus of (N′)yand/or the 3′ terminus of (N)x via a phosphodiester bond. In someembodiments the alkyl moiety comprises propanol, propyl phosphate orpropyl phosphorothioate In some embodiments each of Z and Z′ isindependently selected from propanol, propyl phosphate, propylphosphorothioate combinations thereof or multiples thereof in particular2 or 3 covalently linked propanol, propyl phosphate, propylphosphorothioate or combinations thereof.

In some embodiments each of Z and Z′ is independently selected frompropyl phosphate, propyl phosphorothioate, propyl phospho-propanol;propyl phospho-propyl phosphorothioate; propylphospho-propyl phosphate;(propyl phosphate)3, (propyl phosphate)2-propanol, (propylphosphate)2-propyl phosphorothioate. Any propane or propanol conjugatedmoiety can be included in Z or Z′.

In additional embodiments each of Z and/or Z′ comprises a combination ofan abasic moiety and an unmodified deoxyribonucleotide or ribonucleotideor a combination of a hydrocarbon moiety and an unmodifieddeoxyribonucleotide or ribonucleotide or a combination of an abasicmoiety (deoxyribo or ribo) and a hydrocarbon moiety. In suchembodiments, each of Z and/or Z′ comprises C3Pi-rAb, C3Ps-rAb, C3Ps-dAbor C3Pi-dAb.

According to certain embodiments the invention provides an siRNAcompound further comprising one or more modified ribonucleotide orunconventional moiety, wherein the modified nucleotide possesses amodification in the sugar moiety, in the base moiety or in theinternucleotide linkage moiety. In some embodiments one or more of N orN′ comprises a 2′OMe modified ribonucleotide, a 2′5′ or an L-nucleotide.

In some embodiments (N)x comprises modified and unmodifiedribonucleotides, each modified ribonucleotide having a 2′-O-methyl onits sugar (2′OMe modified or 2′OMe sugar modified), wherein N at the 3′terminus of (N)x is a modified ribonucleotide, (N)x comprises at leastfive alternating modified ribonucleotides beginning at the 3′ end and atleast nine modified ribonucleotides in total and each remaining N is anunmodified ribonucleotide.

In some embodiments at least one of (N)_(x) and (N′)_(y) comprises atleast one mirror nucleotide. In some embodiments in (N′)y at least oneunconventional moiety is present, which unconventional moiety isselected from an abasic ribose moiety, an abasic deoxyribose moiety, amodified or unmodified deoxyribonucleotide, a mirror nucleotide, and anucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotidephosphate bond. In some embodiments at least one of N′ is a mirrornucleotide.

In some embodiments the unconventional moiety is an L-DNA mirrornucleotide. In additional embodiments x=y=19 and at least oneunconventional moiety is present at one of positions 15, 16, 17, or 18in (N′)y. In some embodiments the unconventional moiety is a mirrornucleotide, preferably an L-DNA moiety. In some embodiments the L-DNAmoiety is present at position 17, position 18 or positions 17 and 18.

In some embodiments the unconventional moiety is a nucleotide joined toan adjacent nucleotide by a 2′-5′ internucleotide phosphate bond. Inadditional embodiments x=y=19 and the nucleotides at positions 15-19 or16-19 or 17-19 in (N′)y are joined to adjacent nucleotides by 2′-5′internucleotide phosphate bonds. In some embodiments x=y=19 and thenucleotides at positions 15-19 or 16-19 or 17-19 or 15-18 or 16-18 in(N′)y are joined to the adjacent nucleotides by 2′-5′ internucleotidephosphate bonds.

In some embodiments (N)x comprises nine alternating modifiedribonucleotides. In other embodiments (N)x comprises nine alternatingmodified ribonucleotides further comprising a 2′OMe modified nucleotideat position 2. In some embodiments x=19 and (N)x comprises 2′OMemodified ribonucleotides at the odd numbered positions 1, 3, 5, 7, 9,11, 13, 15, 17, 19. In other embodiments (N)x further comprises a 2′OMemodified ribonucleotide at one or both of positions 2 and 18. In yetother embodiments (N)x comprises 2′OMe modified ribonucleotides atpositions 2, 4, 6, 8, 11, 13, 15, 17, 19. In some embodiments at leastone pyrimidine nucleotide in (N)x comprises a 2′ OMe sugar modification.In some embodiments all pyrimidine nucleotides in (N)x comprises a 2′OMesugar modification. In some embodiments 2, 3, 4, 5, 6, 7, 8, 9, 1′0, 11,12, 13, 14, or 15 pyrimidine nucleotides in N(x) comprise a 2′OMe sugarmodification

In various embodiments z″ is present and is selected from an abasicribose moiety, an abasic deoxyribose moiety, a deoxyribose moiety; aninverted abasic ribose moiety; a C3 moiety, C6-amino-Pi; a mirrornucleotide.

In certain embodiments (N)x is fully complementary to a target sequence.In other embodiments (N)x is substantially complementary to a targetsequence. In some embodiments (N)x comprises one mismatch to the targetsequence. In preferred embodiments (N)x comprises a one-nucleotidemismatch to the target sequence at the 5′ terminus of (N)x; i.e.position 1.

In certain embodiments (N)x and (N′)y are fully complementary. In otherembodiments (N)x and (N′)y are substantially complementary.

In some embodiments (N)x comprises one mismatch to the target sequenceat position 1 and (N)x and (N′)y are fully complementary. In someembodiments (N)x comprises one nucleotide mismatch to the targetsequence at position 1 and (N′)y comprises one nucleotide mismatch to(N)x at position 1.

In some embodiments x=y=19 and in (N)x eighteen consecutive nucleotidesat positions 2-19 are complementary to eighteen consecutive nucleotidesin the target RNA and the nucleotide at position 1 is mismatched to thetarget RNA sequence. In various embodiments the nucleotide at position 1in (N)x is substituted with a moiety selected from the group consistingof ribouracil, modified ribouracil, deoxyribouracil, modifieddeoxyribouracil, pseudouracil, deoxypseudouracil, deoxyribothymidine,modified deoxyribothymidine, ribocytosine, modified ribocytosine,deoxyribocytosine, modified deoxyribocytosine, an abasic ribose moietyand an abasic deoxyribose moiety. In some embodiments the nucleotide atposition 1 in (N)x is substituted with a moiety selected from the groupconsisting of ribouracil, modified ribouracil, deoxyribouracil, modifieddeoxyribouracil.

In some embodiments x=y=19 and in (N)x 18 consecutive nucleotides atpositions 2-19 are complementary to 18 consecutive nucleotides in thetarget RNA and the nucleotide at position 1 is mismatched to the targetRNA sequence and the nucleotide at position 19 of (N′)y is complementaryto the nucleotide at position 1 of (N)x. In other embodiments x=y=19 andthe nucleotide at position 1 of (N)x is mismatched to the target mRNAsequence and the nucleotide at position 19 of (N′)y is mismatched to thenucleotide at position 1 of (N)x.

The double stranded nucleic acid molecules disclosed herein areadvantageous in that they exhibit improved stability and/or improvedactivity and/or reduced off target effects and/or reduced immuneresponse and/or enhanced uptake by cells when compared to blunt ended,or molecules with 3′ dTdT.

Other embodiments are envisaged wherein x=y=21 or wherein x=y=23.Structure (A1) and (A(2) are useful with known and futureoligonucleotide pairs (sense and antisense strands) to a mammalian ornon-mammalian (e.g. viral, bacterial, plant) gene. In some embodimentsthe mammalian gene is a human gene. In various embodiments the mRNA ofthe human gene is set forth in PCT Patent Publication No. WO2009/044392. In additional embodiments the oligonucleotide pair is setforth in PCT Patent Publication No. WO 2009/044392. In furtherembodiments Structures (A1) or (A2) further comprise modifications andmotifs set forth in PCT Patent Publication No. WO 2009/044392.

In another aspect the invention provides a pharmaceutical compositioncomprising a molecule of the invention, in an amount effective toinhibit human gene expression; and a pharmaceutically acceptablecarrier.

More specifically, the invention provides methods and compositionsuseful in treating a subject suffering from acute renal failure (ARF),hearing loss, glaucoma, acute respiratory distress syndrome (ARDS) andother acute lung and respiratory injuries, injury (e.g.ischemia-reperfusion injury) in organ transplant including lung, kidney,bone marrow, heart, pancreas, cornea or liver transplantation andincluding Delayed Graft Function (DGF) nephrotoxicity, spinal cordinjury, pressure sores, dry eye syndrome, oral mucositis, ischemicocular neuropathy (ION) and chronic obstructive pulmonary disease(COPD).

The methods of the invention comprise administering to the subject oneor more siRNA compounds which inhibit expression of a gene. The novelstructures disclosed herein, when integrated into antisense andcorresponding sense nucleic acid sequences to any target gene, providessiRNA compound useful in reducing expression of that target gene. Thetarget gene is a mammalian or non-mammalian gene.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1J show chemical structures of some possible 3′ alkyl/alkylderivative overhangs as covalently attached to the 3′ terminalnucleotide (DNA or RNA) of the oligonucleotide strand via aphosphodiester bond. B on the nucleotide moiety refers to nucleotide“base”; R in each instance is either H or OH; FIG. 1A shows a 3′terminal nucleotide covalently linked to a propanol moiety via aphosphodiester linkage; FIG. 1B shows a 3′ terminal nucleotidecovalently linked via a phosphodiester linkage to a C3Pi moiety; FIG. 1Cshows a 3′ terminal nucleotide covalently linked via a phosphodiesterlinkage to a C3Pi-C3OH moiety (C3 is covalently linked to the C3OH via aphosphosdiester bond); FIG. 1D shows a 3′ terminal nucleotide covalentlylinked to a C3Pi-C3Pi moiety via a phosphodiester linkage; FIG. 1E showsa 3′ terminal nucleotide covalently linked to C3Pi-C3Pi-C3OH via aphosphodiester linkage. FIG. 1F shows a 3′ terminal nucleotidecovalently linked to C3Pi-C3Pi-C3Pi via a phosphodiester linkage. FIG.1G shows a 3′ terminal nucleotide covalently linked to C3Pi-rAb orC3Pi-dAb via a phosphodiester linkage. FIG. 1H shows a 3′ terminalnucleotide covalently linked to rAb-C3Pi (R1=OH) or dAb-C3Pi (R1=H) viaa phosphodiester linkage. FIG. 1J shows a 3′ terminal nucleotidecovalently linked to rAb-rAb (R1=R2=OH) or dAb-rAb (R1=H, R2=OH) orrAb-dAb (R1=OH, R2=H) or dAb-dAb (R1=R2=H) via a phosphodiester linkage.

FIGS. 2A-2F are analogous to FIGS. 1A-1F, except that the 3′-terminusnon-nucleotide overhangs are attached to the 2′ position of the ribosemoiety rather than the 3′-position.

FIGS. 3A-3I illustrate some specific examples of 3′-terminus nucleotideswith overhangs in accordance with embodiments of the invention, in somecases in which an oxygen atom attached to phosphorous has been replacedwith sulfur.

FIG. 4 provides stability data for two double stranded molecules, S505which is a blunt ended 19-mer duplex (compound 5 in Table 3 in theExamples), and S800 which is a 19-mer duplex comprising a non-nucleotideC3C3 3′ terminus overhang (C3Pi-C3OH, compound 7 in Table 3 in theExamples). The two compounds are nuclease stable in cell extract for atleast 36 hours, yet S800 has an IC50 value of about 0.17 nM and S505 hasan IC50 value of 1.1 nM. The sequences used in generating the twocompounds are set forth in SEQ ID NOS:1 and 2 (sense strand andantisense strand, respectively).

DETAILED DESCRIPTION OF THE INVENTION

The present application relates to double stranded siRNA compoundscomprising at least one non-nucleotide moiety covalently attached at the3′ terminus of one or both of the sense and antisense strands. Thenon-nucleotide moiety is selected from an abasic moiety, an invertedabasic moiety, an alkyl moiety or derivative thereof, and an inorganicphosphate.

Structural Design

In one aspect the provided herein are double stranded nucleic acidmolecules comprising a sense strand and an antisense strand, wherein atleast one strand comprises 1, 2, 3, 4, or 5 non-nucleotide moietiescovalently attached at the 3′ terminal end; wherein the non-nucleotidemoiety is selected from an alkyl (hydrocarbon) moiety or a derivativethereof and a phosphate based moiety. In certain preferred embodimentthe non-nucleotide moiety includes an alkyl moiety or an alkylderivative moiety. In some embodiments the at least one strand is theantisense stand. In preferred embodiments the antisense strand comprisestwo non-nucleotide moieties covalently attached at the 3′ terminal end,including C3-C3; C3-C3-Pi; C3-C3-Ps; idAb-idAb

In some embodiments, provided are double stranded nucleic acid moleculeshaving the structure (A1):

(A1) 5′(N)x-Z3′     (antisense strand) 3′Z′-(N′)y-z″5′(sense strand)wherein each of N and N′ is a nucleotide which may be unmodified ormodified, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein at least one of Z or Z′ is present and comprises anon-nucleotide moiety covalently attached at the 3′ terminus of thestrand in which it is present;wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;wherein each of x and y is independently an integer between 18 and 40;wherein the sequence of (N′)y has complementarity to the sequence of(N)x; and wherein the sequence of (N)x has complementarity to aconsecutive sequence in a target RNA.

In some embodiments the covalent bond joining each consecutive N or N′is a phosphodiester bond.

In some embodiments x=y=19 to 27, for example 19, 20, 21, 22, 23, 24,25, 26, 27. In some embodiments x=y and each of x and y is 19, 20, 21,22 or 23. In various embodiments x=y=19.

In some embodiments x=y=19 and one of Z or Z′ is present and consists oftwo non-nucleotide moieties.

In some embodiments x=y=19 and Z′ is present and consists of twonon-nucleotide moieties.

In preferred embodiments x=y=19 and Z is present and consists twonon-nucleotide moieties.

In preferred embodiments x=y=19 and Z is present and consists of twonon-nucleotide moieties; and Z′ is present and consists of onenon-nucleotide moiety.

In additional embodiments x=y=19 and Z and Z′ are present and eachindependently comprises two non-nucleotide moieties.

In some embodiments the double stranded nucleic acid molecules comprisea DNA moiety or a mismatch to the target at position 1 of the antisensestrand (5′ terminus). Such a structure is described herein. According toone embodiment provided are double stranded nucleic acid moleculeshaving a structure (A2) set forth below:

(A2) 5′N1-(N)x-Z3′     (antisense strand)3′Z′-N2-(N′)y-z″5′(sense strand)wherein each of N2, N and N′ is an unmodified or modifiedribonucleotide, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;wherein each of x and y is independently an integer between 17 and 39;wherein the sequence of (N′)y has complementarity to the sequence of(N)x and (N)x has complementarity to a consecutive sequence in a targetRNA;wherein N1 is covalently bound to (N)x and is mismatched to the targetRNA or is a complementary DNA moiety to the target RNA;wherein N1 is a moiety selected from the group consisting of natural ormodified uridine, deoxyribouridine, ribothymidine, deoxyribothymidine,adenosine or deoxyadenosine;wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N2-(N′)y; andwherein at least one of Z or Z′ is present and comprises anon-nucleotide moiety covalently attached at the 3′ terminus of thestrand in which it is present.

In some embodiments x=y=18 and one of Z or Z′ is present and consists oftwo non-nucleotide moieties.

In some embodiments x=y=18 and Z′ is present and consists of twonon-nucleotide moieties.

In preferred embodiments x=y=18 and Z is present and consists twonon-nucleotide moieties.

In preferred embodiments x=y=18 and Z is present and consists of twonon-nucleotide moieties; and Z′ is present and consists of onenon-nucleotide moiety.

In additional embodiments x=y=18 and Z and Z′ are present and eachindependently comprises two non-nucleotide moieties.

In some embodiments the sequence of (N′)y is fully complementary to thesequence of (N)x. In various embodiments sequence of N2-(N′)y iscomplementary to the sequence of N1-(N)x. In some embodiments (N)xcomprises an antisense that is fully complementary to about 17 to about39 consecutive nucleotides in a target RNA. In other embodiments (N)xcomprises an antisense that is substantially complementary to about 17to about 39 consecutive nucleotides in a target RNA.

In some embodiments N1 and N2 form a Watson-Crick base pair. In someembodiments N1 and N2 form a non-Watson-Crick base pair. In someembodiments a base pair is formed between a ribonucleotide and adeoxyribonucleotide.

In some embodiments x=y=18, x=y=19 or x=y=20. In preferred embodimentsx=y=18. When x=18 in N1-(N)x, N1 refers to. position 1 and positions2-19 are included in (N)₁₈ When y=18 in N2-(N′)y, N2 refers to position19 and positions 1-18 are included in (N′)18.

In some embodiments N1 is covalently bound to (N)x and is mismatched tothe target RNA. In various embodiments N1 is covalently bound to (N)xand is a DNA moiety complementary to the target RNA.

In some embodiments a uridine in position 1 of the antisense strand issubstituted with an N1 selected from adenosine, deoxyadenosine,deoxyuridine (dU), ribothymidine or deoxythymidine. In variousembodiments N1 selected from adenosine, deoxyadenosine or deoxyuridine.

In some embodiments guanosine in position 1 of the antisense strand issubstituted with an N1 selected from adenosine, deoxyadenosine, uridine,deoxyuridine, ribothymidine or deoxythymidine. In various embodiments N1is selected from adenosine, deoxyadenosine, uridine or deoxyuridine.

In some embodiments cytidine in position 1 of the antisense strand issubstituted with an N1 selected from adenosine, deoxyadenosine, uridine,deoxyuridine, ribothymidine or deoxythymidine. In various embodiments N1is selected from adenosine, deoxyadenosine, uridine or deoxyuridine.

In some embodiments adenosine in position 1 of the antisense strand issubstituted with an N1 selected from deoxyadenosine, deoxyuridine,ribothymidine or deoxythymidine. In various embodiments N1 selected fromdeoxyadenosine or deoxyuridine.

In some embodiments N1 and N2 form a base pair between uridine ordeoxyuridine, and adenosine or deoxyadenosine. In other embodiments N1and N2 form a base pair between deoxyuridine and adenosine.

In some embodiments the double stranded nucleic acid molecule is asiRNA, siNA or a miRNA. The double stranded nucleic acid molecules asprovided herein are also referred to as “duplexes”.

In certain preferred embodiments x=y=18. In some embodiments N1 and N2form a Watson-Crick base pair. In other embodiments N1 and N2 form anon-Watson-Crick base pair. In certain embodiments N1 is selected fromthe group consisting of riboadenosine, modified riboadenosine,deoxyriboadenosine, modified deoxyriboadenosine. In other embodiments N1is selected from the group consisting of ribouridine, deoxyribouridine,modified ribouridine, and modified deoxyribouridine.

In certain embodiments position 1 in the antisense strand (5′ terminus)comprises deoxyribouridine (dU) or adenosine. In some embodiments N1 isselected from the group consisting of riboadenosine, modifiedriboadenosine, deoxyriboadenosine, modified deoxyriboadenosine and N2 isselected from the group consisting of ribouridine, deoxyribouridine,modified ribouridine, and modified deoxyribouridine. In certainembodiments N1 is selected from the group consisting of riboadenosineand modified riboadenosine and N2 is selected from the group consistingof ribouridine and modified ribouridine.

In certain embodiments N1 is selected from the group consisting ofribouridine, deoxyribouridine, modified ribouridine, and modifieddeoxyribouridine and N2 is selected from the group consisting ofriboadenosine, modified riboadenosine, deoxyriboadenosine, modifieddeoxyriboadenosine. In certain embodiments N1 is selected from the groupconsisting of ribouridine and deoxyribouridine and N2 is selected fromthe group consisting of riboadenosine and modified riboadenosine. Incertain embodiments N1 is ribouridine and N2 is riboadenosine. Incertain embodiments N1 is deoxyribouridine and N2 is riboadenosine.

In some embodiments of Structure (A2), N1 includes 2′ OMe sugar-modifiedribouracil or 2′OMe sugar-modified riboadenosine. In certain embodimentsof structure (A2), N2 includes a 2′OMe sugar modified ribonucleotide ordeoxyribonucleotide.

In some embodiments of Structure (A2), N1 includes 2′ OMe sugar-modifiedribouracil or 2′OMe sugar-modified ribocytosine. In certain embodimentsof structure (A2), N2 includes a 2′OMe sugar modified ribonucleotide.

In some embodiments each of N and N′ is an unmodified nucleotide. Insome embodiments at least one of N or N′ includes a chemically modifiednucleotide or an unconventional moiety. In some embodiments theunconventional moiety is selected from a mirror nucleotide, an abasicribose moiety and an abasic deoxyribose moiety. In some embodiments theunconventional moiety is a mirror nucleotide, preferably an L-DNAmoiety. In some embodiments at least one of N or N′ includes a 2′OMesugar-modified ribonucleotide.

In some embodiments the sequence of (N′)y is fully complementary to thesequence of (N)x. In other embodiments the sequence of (N′)y issubstantially complementary to the sequence of (N)x.

In some embodiments (N)x includes an antisense sequence that is fullycomplementary to about 17 to about 39 consecutive nucleotides in atarget RNA. In other embodiments (N)x includes an antisense that issubstantially complementary to about 17 to about 39 consecutivenucleotides in a target RNA.

In some embodiments the nucleic acid molecules disclosed herein aresiRNA, siNA or miRNA.

In some embodiments of Structures A1 and A2, Z is present and Z′ isabsent. In other embodiments Z′ is present and Z is absent. Inadditional embodiments both Z and Z′ are present. In some embodiments Zand Z′ are present and are identical. In further embodiments Z and Z′are present and are different. In some embodiments Z and Z′ areindependently 2, 3, 4 or 5 non-nucleotide moieties or a combination of2, 3, 4, or 5 non-nucleotide moieties and nucleotides. In someembodiments each of Z and or Z′ consist of two (2) non-nucleotidemoieties covalently attached to the 3′ terminus of the siRNA strand viaa phosphodiester bond.

A non-nucleotide moiety is selected from the group consisting of anabasic moiety, an inverted abasic moiety, an alkyl moiety or derivativethereof, and an inorganic phosphate. In some embodiments anon-nucleotide moiety is an alkyl moiety or derivative thereof. In someembodiments the alkyl moiety comprises a terminal functional groupselected from the group consisting of an alcohol, a terminal amine, aterminal phosphate and a terminal phosphorothioate moiety.

In some embodiments Z is present and comprises one or morenon-nucleotide moieties selected from the group consisting of an abasicmoiety, an inverted abasic moiety, hydrocarbon moiety or derivativethereof, and an inorganic phosphate. In some embodiments Z is presentand consists of two alkyl moieties or derivatives thereof.

In additional embodiments Z′ is present and comprises one or morenon-nucleotide moieties selected from the group consisting of an abasicmoiety, an inverted abasic moiety, a hydrocarbon moiety, and aninorganic phosphate. In some embodiments Z′ is present and comprises oneor more alkyl moieties or derivatives thereof.

In some embodiments Z is present and consists of two alkyl moieties orderivatives thereof and Z′ is present and consists of a single alkylmoiety or derivative thereof.

In some embodiments each of Z and Z′ includes an abasic moiety, forexample a deoxyriboabasic moiety (referred to herein as “dAb”) orriboabasic moiety (referred to herein as “rAb”). In some embodimentseach of Z and/or Z′ comprises two covalently linked abasic moieties andis for example 5′>3′ dAb-dAb or rAb-rAb or dAb-rAb or rAb-dAb. Eachmoiety is covalently conjugated to an adjacent moiety via a covalentbond, preferably a phospho-based bond. In some embodiments thephospho-based bond is a phosphorothioate, a phosphonoacetate or aphosphodiester bond.

In some embodiments each of Z and/or Z′ independently includes a C2, C3,C4, C5 or C6 alkyl moiety, optionally a C3 [propane, —(CH2)3-] moiety ora derivative thereof e.g. propanol (C3-OH), propanediol, orphosphodiester derivative of propanediol (“C3Pi”). In preferredembodiments each of Z and/or Z′ includes two hydrocarbon moieties and insome examples is C3-C3. Each C3 is covalently conjugated to an adjacentC3 via a covalent bond, preferably a phospho-based bond. In someembodiments the phospho-based bond is a phosphorothioate, aphosphonoacetate or a phosphodiester bond.

In some embodiments of Structure A1 and Structure A2 at least one of Zor Z′ is present and comprises at least two non-nucleotide moietiescovalently attached to the strand in which it is present. In someembodiments each of Z and Z′ independently includes a C3 alkyl, C3alcohol or C3 ester moiety. In some embodiments Z′ is absent and Z ispresent and includes a non-nucleotide C3 moiety. In some embodiments Zis absent and Z′ is present and includes a non-nucleotide C3 moiety.

In some embodiments of Structures A1 and A2, each of N and N′ is anunmodified nucleotide. In some embodiments at least one of N or N′includes a chemically modified nucleotide or an unconventional moiety.In some embodiments the unconventional moiety is selected from a mirrornucleotide, an abasic ribose moiety and an abasic deoxyribose moiety. Insome embodiments the unconventional moiety is a mirror nucleotide,preferably an L-DNA moiety. In some embodiments at least one of N or N′includes a 2′OMe sugar-modified ribonucleotide.

In some embodiments the sequence of (N′)y is fully complementary to thesequence of (N)x. In other embodiments the sequence of (N′)y issubstantially complementary to the sequence of (N)x.

In other embodiments the compound of Structure A1 or Structure A2includes at least one ribonucleotide modified in the sugar residue. Insome embodiments the compound includes a modification at the 2′ positionof the sugar residue. In some embodiments the modification in the 2′position includes the presence of an amino, a fluoro, an alkoxy or analkyl moiety. In certain embodiments the 2′ modification includes analkoxy moiety, In preferred embodiments the alkoxy moiety is a methoxymoiety (also known as 2′-O-methyl; 2′OMe; 2′-OCH3). In some embodimentsthe nucleic acid compound includes 2′OMe sugar modified alternatingribonucleotides in one or both of the antisense and the sense strands.In other embodiments the compound includes 2′OMe sugar modifiedribonucleotides in the antisense strand, (N)x or N1-(N)x, only. Incertain embodiments the middle ribonucleotide of the antisense strand;e.g. ribonucleotide in position 10 in a 19-mer strand is unmodified. Invarious embodiments the nucleic acid compound includes at least 5alternating 2′OMe sugar modified and unmodified ribonucleotides. Inadditional embodiments the compound of Structure A1 or Structure A2includes modified ribonucleotides in alternating positions wherein eachribonucleotide at the 5′ and 3′ termini of (N)x or N1-(N)x are modifiedin their sugar residues, and each ribonucleotide at the 5′ and 3′termini of (N′)y or N2-(N)y are unmodified in their sugar residues.

In some embodiments the double stranded molecule includes one or more ofthe following modifications

a) N in at least one of positions 5, 6, 7, 8, or 9 from the 5′ terminusof the antisense strand is selected from a 2′5′ nucleotide or a mirrornucleotide;b) N′ in at least one of positions 9 or 10 from the 5′ terminus of thesense strand is selected from a 2′5′ nucleotide and a pseudoUridine; andc) N′ in 4, 5, or 6 consecutive positions at the 3′ terminus positionsof (N′)y comprises a 2′5′ nucleotide.

In some embodiments the double stranded molecule includes a combinationof the following modifications

a) the antisense strand includes a 2′5′ nucleotide or a mirrornucleotide in at least one of positions 5, 6, 7, 8, or 9 from the 5′terminus; andb) the sense strand includes at least one of a 2′5′ nucleotide and apseudoUridine in positions 9 or 10 from the 5′ terminus.

In some embodiments the double stranded molecule includes a combinationof the following modifications

a) the antisense strand includes a 2′5′ nucleotide or a mirrornucleotide in at least one of positions 5, 6, 7, 8, or 9 from the 5′terminus; andc) the sense strand includes 4, 5, or 6 consecutive 2′5′ nucleotides atthe 3′ penultimate or 3′ terminal positions.

In some embodiments, the sense strand [(N)x or N1-(N)x] includes 1, 2,3, 4, 5, 6, 7, 8, or 9 2′OMe sugar modified ribonucleotides. In someembodiments, the antisense strand includes 2′OMe modifiedribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17 and 19. In otherembodiments antisense strand includes 2′OMe modified ribonucleotides atpositions 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. In other embodiments theantisense strand includes 2′OMe modified ribonucleotides at positions 3,5, 7, 9, 11, 13, 15, 17 and 19. In some embodiments the antisense strandincludes one or more 2′OMe sugar modified pyrimidines. In someembodiments all the pyrimidine nucleotides in the antisense strand are2′OMe sugar modified. In some embodiments the sense strand includes2′OMe sugar modified pyrimidines.

In some embodiments of Structure A1 and Structure A2, the sense strandand the antisense strand are independently phosphorylated orunphosphorylated at the 3′ terminus and at the 5′ terminus. In someembodiments of Structure A1 and Structure A2, the sense strand and theantisense strand are unphosphorylated at the 3′ and 5′ termini. In otherembodiments the sense strand and the antisense strand are phosphorylatedat the 3′ termini.

In some embodiments of Structure A1 and Structure A2 (N)y includes atleast one unconventional moiety selected from a mirror nucleotide, a2′5′ nucleotide and a TNA. In some embodiments the unconventional moietyis a mirror nucleotide. In various embodiments the mirror nucleotide isselected from an L-ribonucleotide (L-RNA) and an L-deoxyribonucleotide(L-DNA). In preferred embodiments the mirror nucleotide is L-DNA. Incertain embodiments the sense strand comprises an unconventional moietyin position 9 or 10 (from the 5′ terminus). In preferred embodiments thesense strand includes an unconventional moiety in position 9 (from the5′ terminus). In some embodiments the sense strand is 19 nucleotides inlength and comprises 4, 5, or 6 consecutive unconventional moieties inpositions 15, (from the 5′ terminus). In some embodiments the sensestrand includes 4 consecutive 2′5′ ribonucleotides in positions 15, 16,17, and 18. In some embodiments the sense strand includes 5 consecutive2′5′ ribonucleotides in positions 15, 16, 17, 18 and 19. In variousembodiments the sense strand further comprises Z′. In some embodimentsZ′ includes a C3OH moiety or a C3Pi moiety.

In some embodiments of Structure A1 (N′)y includes at least one L-DNAmoiety. In some embodiments x=y=19 and (N′)y, consists of unmodifiedribonucleotides at positions 1-17 and 19 and one L-DNA at the 3′penultimate position (position 18). In other embodiments x=y=19 and(N′)y consists of unmodified ribonucleotides at positions 1-16 and 19and two consecutive L-DNA at the 3′ penultimate position (positions 17and 18). In various embodiments the unconventional moiety is anucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotidephosphate linkage. According to various embodiments (N′)y includes 2, 3,4, 5, or 6 consecutive ribonucleotides at the 3′ terminus linked by2′-5′ internucleotide linkages. In one embodiment, four consecutivenucleotides at the 3′ terminus of (N′)y are joined by three 2′-5′phosphodiester bonds, wherein one or more of the 2′-5′ nucleotides whichform the 2′-5′ phosphodiester bonds further includes a 3′-O-methyl(3′OMe) sugar modification. Preferably the 3′ terminal nucleotide of(N′)y includes a 2′OMe sugar modification. In certain embodiments x=y=19and (N′)y includes two or more consecutive nucleotides at positions 15,16, 17, 18 and 19 include a nucleotide joined to an adjacent nucleotideby a 2′-5′ internucleotide bond (2′-5′ nucleotide). In variousembodiments the nucleotide forming the 2′-5′ internucleotide bondincludes a 3′ deoxyribose nucleotide or a 3′ methoxy nucleotide (3′ H or3′OMe in place of a 3′ OH). In some embodiments x=y=19 and (N′)yincludes 2′-5′ nucleotides at positions 15, 16 and 17 such that adjacentnucleotides are linked by a 2′-5′ internucleotide bond between positions15-16, 16-17 and 17-18; or at positions, 15, 16, 17, 18, and 19 suchthat adjacent nucleotides are linked by a 2′-5′ internucleotide bondbetween positions 15-16, 16-17, 17-18 and 18-19 and a 3′OH is availableat the 3′ terminal nucleotide or at positions 16, 17 and 18 such thatadjacent nucleotides are linked by a 2′-5′ internucleotide bond betweenpositions 16-17, 17-18 and 18-19. In some embodiments x=y=19 and (N′)yincludes 2′-5′ nucleotides at positions 16 and 17 or at positions 17 and18 or at positions 15 and 17 such that adjacent nucleotides are linkedby a 2′-5′ internucleotide bond between positions 16-17 and 17-18 orbetween positions 17-18 and 18-19 or between positions 15-16 and 17-18,respectively. In other embodiments the pyrimidine ribonucleotides (rU,rC) in (N′)y are substituted with nucleotides joined to the adjacentnucleotide by a 2′-5′ internucleotide bond. In some embodiments x=y=19and (N′)y comprises five consecutive nucleotides at the 3′ terminusjoined by four 2′-5′ linkages, specifically the linkages between thenucleotides position 15-16, 16-17, 17-18 and 18-19.

In some embodiments x=y=19 and (N′)y comprises five consecutivenucleotides at the 3′ terminus joined by four 2′-5′ linkages andoptionally further includes Z′ and z′ independently selected from aninverted abasic moiety and a C3 alkyl [C3; 1,3-propanediolmono(dihydrogen phosphate)] cap. The C3 alkyl cap is covalently linkedto the 3′ or 5′ terminal nucleotide. In some embodiments the 3′ C3terminal cap further comprises a 3′ phosphate. In some embodiments the3′ C3 terminal cap further comprises a 3′ terminal hydroxyl group.

In some embodiments x=y=19 and (N′)y comprises an L-DNA position 18; and(N′)y optionally further includes Z′ and z′ independently selected froman inverted abasic moiety and a C3 alkyl [C3; 1,3-propanediolmono(dihydrogen phosphate)] cap.

In some embodiments (N′)y comprises a 3′ terminal phosphate (i.e.phosphorylated at the 3′ terminus). In some embodiments (N′)y comprisesa 3′ terminal hydroxyl.

In some embodiments x=y=19 and (N)x includes 2′OMe sugar modifiedribonucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or atpositions 2, 4, 6, 8, 11, 13, 15, 17, 19. In some embodiments x=y=19 and(N)x includes 2′OMe sugar modified pyrimidines. In some embodiments allpyrimidines in (N)x include the 2′OMe sugar modification.

In some embodiments of structure A2 x=y=18 and N2 is a riboadenosinemoiety. In some embodiments x=y=18, and N2-(N′)y comprises fiveconsecutive nucleotides at the 3′ terminus joined by four 2′-5′linkages, specifically the linkages between the nucleotides position15-16, 16-17, 17-18 and 18-19. In some embodiments the linkages includephosphodiester bonds. In some embodiments x=y=18 and N2-(N′)y comprisesfive consecutive nucleotides at the 3′ terminus joined by four 2′-5′linkages and optionally further includes Z′ and z′ independentlyselected from an inverted abasic moiety and a C3 alkyl [C3;1,3-propanediol mono(dihydrogen phosphate)] cap. In some embodimentsx=y=18 and N2-(N′)y comprises an L-DNA position 18; and (N′)y optionallyfurther includes Z′ and z′ independently selected from an invertedabasic moiety and a C3 alkyl [C3; 1,3-propanediol mono(dihydrogenphosphate)] cap. In some embodiments N2-(N′)y comprises a 3′ terminalphosphate. In some embodiments N2-(N′)y comprises a 3′ terminalhydroxyl. In some embodiments x=y=18 and N1-(N)x includes 2′OMe sugarmodified ribonucleotides in positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19or in positions 1, 3, 5, 9, 11, 13, 15, 17, 19, or in positions 3, 5, 9,11, 13, 15, 17, or in positions 2, 4, 6, 8, 11, 13, 15, 17, 19. In someembodiments x=y=18 and N1-(N)x includes 2′OMe sugar modifiedribonucleotides at positions 11, 13, 15, 17 and 19 (from 5′ terminus).In some embodiments x=y=18 and N1-(N)x includes 2′OMe sugar modifiedribonucleotides in positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or inpositions 3, 5, 7, 9, 11, 13, 15, 17, 19. In some embodiments x=y=18 andN1-(N)x includes 2′OMe sugar modified ribonucleotides in positions 2, 4,6, 8, 11, 13, 15, 17, 19.

In some embodiments x=y=18 and N1-(N)x includes 2′OMe sugar modifiedpyrimidines. In some embodiments all pyrimidines in (N)x include the2′OMe sugar modification. In some embodiments the antisense strandfurther comprises an L-DNA or a 2′-5′ nucleotide in position 5, 6 or 7(5′>3′). In other embodiments the antisense strand further comprises aribonucleotide, which generates a 2′5′ internucleotide linkage inbetween the ribonucleotides in positions 5-6 or 6-7 (5′>3′).

In additional embodiments N1-(N)x further includes Z wherein Z comprisesa non-nucleotide overhang. In some embodiments the non-nucleotideoverhang is C3-C3 [1,3-propanediol mono(dihydrogen phosphate)]2.

In some embodiments of Structure A2, (N)y includes at least one L-DNAmoiety. In some embodiments x=y=18 and (N′)y consists of unmodifiedribonucleotides at positions 1-16 and 18 and one L-DNA at the 3′penultimate position (position 17). In other embodiments x=y=18 and(N′)y consists of unmodified ribonucleotides at position 1-15 and 18 andtwo consecutive L-DNA at the 3′ penultimate position (positions 16 and17). In various embodiments the unconventional moiety is a nucleotidejoined to an adjacent nucleotide by a 2′-5′ internucleotide phosphatelinkage. According to various embodiments (N′)y includes 2, 3, 4, 5, or6 consecutive ribonucleotides at the 3′ terminus linked by 2′-5′internucleotide linkages. In one embodiment, four consecutivenucleotides at the 3′ terminus of (N′)y are joined by three 2′-5′phosphodiester bonds, wherein one or more of the 2′-5′ nucleotides whichform the 2′-5′ phosphodiester bonds further includes a 3′-O-methyl(3′OMe) sugar modification. Preferably the 3′ terminal nucleotide of(N′)y includes a 2′OMe sugar modification. In certain embodiments x=y=18and in (N′)y two or more consecutive nucleotides at positions 14, 15,16, 17, and 18 include a nucleotide joined to an adjacent nucleotide bya 2′-5′ internucleotide bond. In various embodiments the nucleotideforming the 2′-5′ internucleotide bond includes a 3′ deoxyribosenucleotide or a 3′ methoxy nucleotide. In some embodiments x=y=18 and(N′)y includes nucleotides joined to the adjacent nucleotide by a 2′-5′internucleotide bond between positions 15-16, 16-17 and 17-18 or betweenpositions 16-17 and 17-18. In some embodiments x=y=18 and (N′)y includesnucleotides joined to the adjacent nucleotide by a 2′-5′ internucleotidebond between positions 14-15, 15-16, 16-17, and 17-18 or betweenpositions 15-16, 16-17, and 17-18 or between positions 16-17 and 17-18or between positions 17-18 or between positions 15-16 and 17-18. Inother embodiments the pyrimidine ribonucleotides (rU, rC) in (N′)y aresubstituted with nucleotides joined to the adjacent nucleotide by a2′-5′ internucleotide bond.

In some embodiments of Structure A1 and Structure A2 each N consists ofan unmodified ribonucleotide. In some embodiments of Structure A1 andStructure A2 each N′ consists of an unmodified nucleotide. In preferredembodiments, at least one of N and N′ is a modified ribonucleotide or anunconventional moiety.

In other embodiments the molecule of Structure A1 or Structure A2includes at least one ribonucleotide modified in the sugar residue. Insome embodiments the compound includes a modification at the 2′ positionof the sugar residue. In some embodiments the modification at the 2′position includes the presence of an amino, a fluoro, an alkoxy or analkyl moiety. In certain embodiments the 2′ modification includes analkoxy moiety, In preferred embodiments the alkoxy moiety is a methoxymoiety (also known as 2′-O-methyl; 2′OMe; 2′-OCH3). In some embodimentsthe nucleic acid compound includes 2′OMe sugar modified alternatingribonucleotides in one or both of the antisense and the sense strands.In other embodiments the compound includes 2′OMe sugar modifiedribonucleotides in the antisense strand, (N)x or N1-(N)x, only. Incertain embodiments the middle ribonucleotide of the antisense strand;e.g. ribonucleotide in position 10 in a 19-mer strand is unmodified. Invarious embodiments the nucleic acid compound includes at least 5alternating 2′OMe sugar modified and unmodified ribonucleotides.

In additional embodiments the compound of Structure A1 or Structure A2includes modified ribonucleotides in alternating positions wherein eachribonucleotide at the 5′ and 3′ termini of (N)x or N1-(N)x are modifiedin their sugar residues, and each ribonucleotide at the 5′ and 3′termini of (N′)y or N2-(N)y are unmodified in their sugar residues.

In some embodiments, (N)x or N1-(N)x includes 2′OMe modifiedribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17 and 19. In otherembodiments (N)x (N)x or N1-(N)x includes 2′OMe modified ribonucleotidesat positions 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. In some embodiments(N)x or N1-(N)x includes 2′OMe modified pyrimidines. In some embodimentsall the pyrimidine nucleotides in (N)x or N1-(N)x are 2′OMe modified. Insome embodiments (N′)y or N2-(N′)y includes 2′OMe modified pyrimidines.In additional embodiments the compound of Structure A1 or Structure A2includes modified ribonucleotides in alternating positions wherein eachribonucleotide at the 5′ and 3′ termini of (N)x or N1-(N)x are modifiedin their sugar residues, and each ribonucleotide at the 5′ and 3′termini of (N′)y or N2-(N)y are unmodified in their sugar residues.

The nucleic acid molecules disclosed herein may have a blunt end on oneend, for example when Z and z″ are absent or wherein Z′ is absent. Thenucleic acid molecule may be modified with modified nucleotides orunconventional moieties that may be located at any position along eitherthe sense or antisense strand. The nucleic acid molecule may includeabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 modifiednucleotides. The nucleic acid molecule may include about 1, 2, 3, 4, 5,6, 7, or 8 unconventional moieties. The nucleic acid molecule mayinclude a group of about 1, 2, 3, 4, 5, 6, 7, or 8, preferably 1, 2, 3or 4 contiguous modified nucleotides or unconventional moieties.Modified nucleic acids may be present in the sense strand only, theantisense strand only, or in both the sense strand and the antisensestrand. In some embodiments the modified nucleotide comprises a 2′ sugarmodified nucleotide, including 2′O-methyl modified nucleotide, 2′deoxyfluoro modified nucleotide, 2′-amino modified nucleotide. In someembodiments the unconventional moiety comprises a mirror nucleotide(i.e. L-DNA or L-RNA) or a nucleotide able to form a 2′-5′ linkage (2′5′nucleotide).

As used herein, the term “duplex region” refers to the region in thedouble stranded molecule in which two complementary or substantiallycomplementary oligonucleotides form base pairs with one another,typically by Watson-Crick base pairing or by any other manner thatallows for a duplex formation. For example, an oligonucleotide strandhaving 19 nucleotide units can base pair with a complementaryoligonucleotide of 19 nucleotide units, or can base pair with 15, 16 17or 18 bases on each strand such that the “duplex region” consists of 15,16 17 or 18 base pairs. The remaining base pairs may, for example, existas 5′ and 3′ overhangs. Further, within the duplex region, 100%complementarity is not required; substantial complementarity isallowable within a duplex region. The overhang region may consist ofnucleotide or non-nucleotide moieties. As disclosed herein at least oneoverhang region consists of one or more non-nucleotide moieties.

Generic non-limiting nucleic acid molecule patterns are shown belowwhere N′=sense strand nucleotide in the duplex region; z″=5′-cappingmoiety covalently attached at the 5′ terminus of the sense strand; C3=3carbon non-nucleotide moiety; N=antisense strand nucleotide in theduplex region; idB=inverted abasic deoxyribonucleotide non-nucleotidemoiety. Each N, N′, is independently modified or unmodified or anunconventional moiety. The sense and antisense strands are eachindependently 18-40 nucleotides in length. The examples provided belowhave a duplex region of 19 nucleotides; however, nucleic acid moleculesdisclosed herein can have a duplex region anywhere between 18 and 40nucleotides and where each strand is independently between 18 and 40nucleotides in length. In each duplex the antisense strand (N)x is shownon top. In some embodiments a double stranded nucleic acid molecule hasthe following structure:

5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Pi3′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Pi3′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Pi3′PiC3-PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Pi3′PiC3-PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′ N′-z″5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Pi3′PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Pi3′PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Pi3′HOC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Pi3′HOC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″5′N N N N N N N N N N N N N N N N N N N-aB-aB3′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′5′N N N N N N N N N N N N N N N N N N N-aB-aB3′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″5′N N N N N N N N N N N N N N N N N N N-idB-idB3′aB-aB-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′5′N N N N N N N N N N N N N N N N N N N-aB-aB3′aB-aB-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Pi3′PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Pi3′aB-aB-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′PiC3-PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′PiC3-PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′ N′-z″5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′HOC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′HOC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Ps3′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Ps3′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Ps3′OHC3-PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3Ps3′OHC3-PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′ N′-z″

In some preferred embodiment nucleic acid molecules disclosed hereinhave the following structure

5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′HOC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″ or5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′iPC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″wherein of N and N′ is independently a ribonucleotide which may beunmodified or modified, or is an unconventional moiety;wherein each N is linked to the adjacent N by a covalent bond;wherein each N′ is linked to the adjacent N′ by a covalent bond; andwherein z″ is a capping moiety covalently attached to the 5′ terminus ofthe sense strand. The term “aB” refers to an abasic moiety which can beriboabasic moiety or a deoxyriboabasic moiety, or an inverted riboabasicmoiety or an inverted deoxyriboabasic moiety.

In some embodiments the nucleic acid molecules disclosed herein compriseZ. In other embodiments the nucleic acid molecules disclosed hereincomprise Z′. In additional embodiments both Z and Z′ are present. Insome embodiments Z and Z′ are both present and identical. In furtherembodiments both Z and Z′ are present and are different. In someembodiments Z and Z′ independently comprise 1 or 2 non-nucleotidemoieties. In some embodiments Z and Z′ independently comprise 2non-nucleotide moieties.

In some embodiments Z is present and comprises one or morenon-nucleotide moieties selected from an abasic moiety an invertedabasic moiety, an alkyl moiety or derivative thereof, and an inorganicphosphate moiety.

In additional embodiments Z′ is present and comprises one or morenon-nucleotide moieties selected from an abasic moiety an invertedabasic moiety, an alkyl moiety or derivative thereof or an inorganicphosphate moiety.

In additional embodiments Z and/or Z′ are present and independentlycomprise a combination of one or more nucleotide and one or morenon-nucleotide moiety selected from the moieties disclosed herein.

In some embodiments each of Z and Z′ includes an abasic moiety,optionally deoxyriboabasic (referred to herein as “dAb”) or riboabasic(referred to herein as “rAb”) nucleotides. In some embodiments each of Zand/or Z′ is dAb-dAb or rAb-rAb.

In some embodiments each of Z and/or Z′ independently includes an alkylmoiety, optionally a phosphodiester derivative of propanediol((CH2)3-Pi, referred to herein also as “C3Pi”) modified moiety. In someembodiments Z and/or Z′ are C3Pi-C3Pi. In a specific embodiment x=y=19and Z comprises two propanediol derivatives, C3-C3 (i.e. —C3-Pi-C3-Pi).In various embodiments the C3 moiety is covalently linked to the 3′terminus of the sense or antisense strand via a phosphodiester bond.

In additional embodiments Z and/or Z′ comprise a combination of one ormore abasic moieties and unmodified nucleotides or a combination of oneor more hydrocarbon moieties and unmodified nucleotides or a combinationof one or more abasic and hydrocarbon moieties. In such embodiments, Zand/or Z′ are optionally C3-rAb or C3-dAb.

In further embodiments relating to structure A1 or A2, the nucleic acidmolecules further comprises a 2′O-Me modification on the sugar ofribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17 and 19 of theantisense strand. In additional embodiments the compound also comprisesan L-DNA nucleotide at position 18 of the sense strand. In additionalembodiments the compound comprises a nucleotide joined to an adjacentnucleotide by a 2′-5′ internucleotide phosphate bond. In additionalembodiments x=y=19 and the nucleotides at positions 15-19 or 16-19 or17-19 in (N′)y are joined to adjacent nucleotides by 2′-5′internucleotide phosphate bonds. In some embodiments x=y=19 and thenucleotides at positions 15-19 or 16-19 or 17-19 or 15-18 or 16-18 in(N′)y are joined to the adjacent nucleotides by 2′-5′ internucleotidephosphate bonds.

According to certain embodiments the invention provides an siRNAcompound further comprising one or more modified nucleotide, wherein themodified nucleotide possesses a modification in the sugar moiety, in thebase moiety or in the internucleotide linkage moiety.

In some embodiments (N)x comprises modified and unmodifiedribonucleotides, each modified ribonucleotide having a 2′-O-methyl onits sugar, wherein N at the 3′ terminus of (N)x is a modifiedribonucleotide, (N)x comprises at least five alternating modifiedribonucleotides beginning at the 3′ end and at least nine modifiedribonucleotides in total and each remaining N is an unmodifiedribonucleotide.

In some embodiments at least one of (N)_(x) and (N′)_(y) comprises atleast one mirror nucleotide. In some embodiments in (N′)y at least oneunconventional moiety is present, which unconventional moiety may be anabasic ribose moiety, an abasic deoxyribose moiety, a modified orunmodified deoxyribonucleotide, a mirror nucleotide, and a nucleotidejoined to an adjacent nucleotide by a 2′-5′ internucleotide phosphatebond, or any other unconventional moiety disclosed herein.

In some embodiments an unconventional moiety is an L-DNA mirrornucleotide; in additional embodiments at least one unconventional moietyis present at positions 15, 16, 17, or 18 in (N′)y. In some embodimentsthe unconventional moiety is selected from a mirror nucleotide, anabasic ribose moiety and an abasic deoxyribose moiety. In someembodiments the unconventional moiety is a mirror nucleotide, preferablyan L-DNA moiety. In some embodiments the L-DNA moiety is present atposition 17, position 18 or positions 17 and 18.

In yet other embodiments (N′)y comprises at least five abasic ribosemoieties or abasic deoxyribose moieties and at least one of N′ is anLNA.

In some embodiments (N)x comprises nine alternating modifiedribonucleotides. In other embodiments (N)x comprises nine alternatingmodified ribonucleotides further comprising a 2′ modified nucleotide atposition 2. In some embodiments (N)x comprises 2′OMe modifiedribonucleotides at the odd numbered positions 1, 3, 5, 7, 9, 11, 13, 15,17, 19. In other embodiments (N)x further comprises a 2′OMe modifiedribonucleotide at one or both of positions 2 and 18. In yet otherembodiments (N)x comprises 2′OMe modified ribonucleotides at positions2, 4, 6, 8, 11, 13, 15, 17, 19. In some embodiments at least onepyrimidine nucleotide in (N)x comprises a 2′OMe sugar modification. Insome embodiments all pyrimidine nucleotides in (N)x comprises a 2′OMesugar modification. In some embodiments 2, 3, 4, 5, 6, 7, 8, 9, 1′0, 11,12, 13, 14, or 15 pyrimidine nucleotides in N(x) comprise a 2′OMe sugarmodification

In various embodiments z″ is present and is selected from an abasicribose moiety, a deoxyribose moiety; an inverted abasic ribose moiety, adeoxyribose moiety; C6-amino-Pi; a mirror nucleotide.

In one embodiment of the nucleic acid molecules (N′)y comprises at leasttwo nucleotides at either or both the 5′ and 3′ termini of (N′)y arejoined by a 2′-5′ phosphodiester bond. In certain embodiments x=y=19; in(N)x the nucleotides alternate between modified ribonucleotides andunmodified ribonucleotides, each modified ribonucleotide being modifiedso as to have a 2′-O-methyl on its sugar and the ribonucleotide locatedat the middle of (N)x being unmodified; and three nucleotides at the 3′terminus of (N′)y are joined by two 2′-5′ phosphodiester bonds. In otherembodiments, x=y=19; in (N)x the nucleotides alternate between modifiedribonucleotides and unmodified ribonucleotides, each modifiedribonucleotide being modified so as to have a 2′-O-methyl on its sugarand the ribonucleotide located at the middle of (N)x being unmodified;and four consecutive nucleotides at the 5′ terminus of (N′)y are joinedby three 2′-5′ phosphodiester bonds. In a further embodiment, anadditional nucleotide located in the middle position of (N)y may bemodified with 2′-O-methyl on its sugar. In another embodiment, in (N)xthe nucleotides alternate between 2′-O-methyl modified ribonucleotidesand unmodified ribonucleotides, and in (N′)y four consecutivenucleotides at the 5′ terminus are joined by three 2′-5′ phosphodiesterbonds and the 5′ terminal nucleotide or two or three consecutivenucleotides at the 5′ terminus comprise 3′-O-Me sugar modifications.

In certain embodiments of Structure (A1), x=y=19 and in (N′)y thenucleotide in at least one position comprises a mirror nucleotide, adeoxyribonucleotide and a nucleotide joined to an adjacent nucleotide bya 2′-5′ internucleotide bond.

In certain embodiments of Structure (A1), x=y=19 and (N′)y comprises amirror nucleotide. In various embodiments the mirror nucleotide is anL-DNA nucleotide. In certain embodiments the L-DNA isL-deoxyribocytidine. In some embodiments (N′)y comprises L-DNA atposition 18. In other embodiments (N′)y comprises L-DNA at positions 17and 18. In certain embodiments (N′)y comprises L-DNA substitutions atpositions 2 and at one or both of positions 17 and 18. Other embodimentsof Structure (A1) are envisaged in wherein x=y=21 or wherein x=y=23; inthese embodiments the modifications for (N′)y discussed above instead ofbeing on positions 15, 16, 17, 18 are on positions 17, 18, 19, 20 for 21mer and on positions 19, 20, 21, 22 for 23 mer; similarly themodifications at one or both of positions 17 and 18 are on one or bothof positions 19 or 20 for the 21 mer and one or both of positions 21 and22 for the 23 mer. All modifications in the 19 mer are similarlyadjusted for the 21 and 23 mers.

According to various embodiments of Structure A1 or A2 in 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides at the 3′terminus in (N′)y or N2-(N′)y are linked by 2′-5′ internucleotidelinkages In one embodiment, four consecutive nucleotides at the 3′terminus of (N′)y are joined by three 2′-5′ phosphodiester bonds,wherein one or more of the 2′-5′ nucleotides which form the 2′-5′phosphodiester bonds further comprises a 3′-O-methyl sugar modification.Preferably the 3′ terminal nucleotide of (N′)y comprises a 2′-O-methylsugar modification. In certain embodiments of Structure (A1), x=y=19 andin (N′)y two or more consecutive nucleotides at positions 15, 16, 17, 18and 19 comprise a nucleotide joined to an adjacent nucleotide by a 2′-5′internucleotide bond. In various embodiments the nucleotide forming the2′-5′ internucleotide bond comprises a 3′ deoxyribose nucleotide or a 3′methoxy nucleotide. In some embodiments the nucleotides at positions 17and 18 in (N′)y are joined by a 2′-5′ internucleotide bond. In otherembodiments the nucleotides at positions 16, 17, 18, 16-17, 17-18, or16-18 in (N′)y are joined by a 2′-5′ internucleotide bond.

In certain embodiments (N′)y comprises an L-DNA at position 2 and 2′-5′internucleotide bonds at positions 16, 17, 18, 16-17, 17-18, or 16-18.In certain embodiments (N′)y comprises 2′-5′ internucleotide bonds atpositions 16, 17, 18, 16-17, 17-18, or 16-18 and a 5′ terminal capnucleotide.

In one embodiment of the nucleic acid molecules, the 3′ terminalnucleotide or two or three consecutive nucleotides at the 3′ terminus of(N′)y are L-deoxyribonucleotides.

In other embodiments the nucleic acid molecules, in (N′)y 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides at eitherterminus or 2-8 modified nucleotides at each of the 5′ and 3′ terminiare independently 2′ sugar modified nucleotides. In some embodiments the2′ sugar modification comprises the presence of an amino, a fluoro, analkoxy or an alkyl moiety. In certain embodiments the 2′ sugarmodification comprises a methoxy moiety (2′-OMe).

In one embodiment, three, four or five consecutive nucleotides at the 5′terminus of (N′)y comprise the 2′-OMe modification. In anotherembodiment, three consecutive nucleotides at the 3′ terminus of (N′)ycomprise the 2′-O-Me sugar modification.

In some embodiments of Structure A1 or A2 in (N′)y or N2-(N′)y 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides ateither or 2-8 modified nucleotides at each of the 5′ and 3′ termini areindependently bicyclic nucleotide. In various embodiments the bicyclicnucleotide is a locked nucleic acid (LNA). A 2′-0, 4′-C-ethylene-bridgednucleic acid (ENA) is a species of LNA (see below).

In various embodiments (N′)y or N2-(N′)y comprises modified nucleotidesat the 5′ terminus or at both the 3′ and 5′ termini.

In some embodiments of Structure A1 or A2 at least two nucleotides ateither or both the 5′ and 3′ termini of (N′)y are joined by P-ethoxybackbone modifications. In certain embodiments x=y=19 or x=y=23; in (N)xthe nucleotides alternate between modified ribonucleotides andunmodified ribonucleotides, each modified ribonucleotide being modifiedso as to have a 2′-O-methyl on its sugar and the ribonucleotide locatedat the middle position of (N)x being unmodified; and four consecutivenucleotides at the 3′ terminus or at the 5′ terminus of (N′)y are joinedby three P-ethoxy backbone modifications. In another embodiment, threeconsecutive nucleotides at the 3′ terminus or at the 5′ terminus of(N′)y are joined by two P-ethoxy backbone modifications.

In some embodiments of Structure A1 or A2 in (N′)y or N2-(N′)y 2, 3, 4,5, 6, 7 or 8, consecutive ribonucleotides at each of the 5′ and 3′termini are independently mirror nucleotides, nucleotides joined by2′-5′ phosphodiester bond, 2′ sugar modified nucleotides or bicyclicnucleotide. In one embodiment, the modification at the 5′ and 3′ terminiof (N′)y is identical. In one embodiment, four consecutive nucleotidesat the 5′ terminus of (N′)y are joined by three 2′-5′ phosphodiesterbonds and three consecutive nucleotides at the 3′ terminus of (N′)y arejoined by two 2′-5′ phosphodiester bonds. In another embodiment, themodification at the 5′ terminus of (N′)y is different from themodification at the 3′ terminus of (N′)y. In one embodiment, themodified nucleotides at the 5′ terminus of (N′)y are mirror nucleotidesand the modified nucleotides at the 3′ terminus of (N′)y are joined by2′-5′ phosphodiester bond. In another specific embodiment, threeconsecutive nucleotides at the 5′ terminus of (N′)y are LNA nucleotidesand three consecutive nucleotides at the 3′ terminus of (N′)y are joinedby two 2′-5′ phosphodiester bonds. In (N)x the nucleotides alternatebetween modified ribonucleotides and unmodified ribonucleotides, eachmodified ribonucleotide being modified so as to have a 2′-O-methyl onits sugar and the ribonucleotide located at the middle of (N)x beingunmodified, or the ribonucleotides in (N)x being unmodified

In another embodiment of Structure A1 the invention provides a compoundwherein x=y=19; in (N)x the nucleotides alternate between modifiedribonucleotides and unmodified ribonucleotides, each modifiedribonucleotide being modified so as to have a 2′-O-methyl on its sugarand the ribonucleotide located at the middle of (N)x being unmodified;three nucleotides at the 3′ terminus of (N′)y are joined by two 2′-5′phosphodiester bonds and three nucleotides at the 5′ terminus of (N′)yare LNA such as ENA; and Z and/or Z′ independently comprise one or morenon-nucleotide moiety selected from the group consisting of an abasicmoiety, an inverted abasic moiety, a hydrocarbon moiety, and aninorganic phosphate, or a combination of one or more non-nucleotidemoiety and one or more nucleotide. In some embodiments Z is selectedfrom C3Pi-C3Pi, C3Pi-C3OH; C3Pi-rAb; C3Pi-dAb; dAb-dAb and rAb-rAb.

In another embodiment five consecutive nucleotides at the 5′ terminus of(N′)y or N2-(N′)y comprise the 2′-O-methyl sugar modification and twoconsecutive nucleotides at the 3′ terminus of (N′)y are L-DNA.

According to other embodiments in N′)y or N2-(N′)y the 5′ or 3′ terminalnucleotide, or 2, 3, 4, 5 or 6 consecutive nucleotides at either terminior 1-4 modified nucleotides at each of the 5′ and 3′ termini areindependently phosphonocarboxylate or phosphinocarboxylate nucleotides(PACE nucleotides). In some embodiments the PACE nucleotides aredeoxyribonucleotides. In some embodiments in N′)y or N2-(N′)y, 1 or 2consecutive nucleotides at each of the 5′ and 3′ termini are PACEnucleotides. Examples of PACE nucleotides and analogs are disclosed inU.S. Pat. Nos. 6,693,187 and 7,067,641 both incorporated by reference.

In one embodiment of Structure (A1), x=y=19; (N)x comprises unmodifiedribonucleotides in which two consecutive nucleotides linked by one 2′-5′internucleotide linkage at the 3′ terminus; (N′)y comprises unmodifiedribonucleotides in which two consecutive nucleotides linked by one 2′-5′internucleotide linkage at the 5′ terminus; and Z and/or Z′independently comprise one or more non-nucleotide moiety selected fromthe group consisting of an abasic moiety, an inverted abasic moiety, ahydrocarbon moiety, and an inorganic phosphate, or a combination of oneor more non-nucleotide moiety and one or more nucleotide. In someembodiments Z is selected from C3Pi-C3Ps; C3Pi-C3OH; C3Pi-C3Pi;C3Pi-rAb; C3Pi-dAb; dAb-dAb and rAb-rAb, each C3, rAb, dAb covalentlylinked to the adjacent C3Pi, rAb, dAb via a phospho-based bond. In someembodiments the phospho-based bond is a phosphodiester bond or aphosphorothiophosphate bond.

In some embodiments, x=y=19; (N)x comprises unmodified ribonucleotidesin which three consecutive nucleotides at the 3′ terminus are joinedtogether by two 2′-5′ phosphodiester bonds; (N′)y comprises unmodifiedribonucleotides in which four consecutive nucleotides at the 5′ terminusare joined together by three 2′-5′ phosphodiester bonds; and. Z and/orZ′ independently comprise one or more non-nucleotide moiety selectedfrom the group consisting of an abasic moiety, an inverted abasicmoiety, a hydrocarbon moiety, and an inorganic phosphate, or acombination of one or more non-nucleotide moiety and one or morenucleotide. In some embodiments Z is selected from C3Pi-C3Ps; C3Pi-C3OH;C3Pi-C3Pi; C3Pi-rAb; C3Pi-dAb; dAb-dAb and rAb-rAb wherein each C3Pi,rAb, dAb covalently linked to the adjacent C3Pi, rAb, dAb via aphospho-based bond. In some embodiments the phospho-based bond is aphosphodiester bond or a phosphorothiophosphate bond.

According to one embodiment of Structure A1 or A2 four consecutivenucleotides at the 5′ terminus of (N′)y or (N′)y-N2, respectively arejoined by three 2′-5′ phosphodiester bonds; three consecutivenucleotides at the 3′ terminus of (N′)x are joined by two 2′-5′phosphodiester bonds; and Z and/or Z′ independently comprise one or morenon-nucleotide moiety selected from the group consisting of an abasicmoiety, an inverted abasic moiety, a hydrocarbon moiety, and aninorganic phosphate, or a combination of one or more non-nucleotidemoiety and one or more nucleotide. In some embodiments Z is selectedfrom C3Pi-C3Ps; C3Pi-C3OH; C3Pi-C3Pi; C3Pi-rAb; C3Pi-dAb; C3-dAb;dAb-dAb and rAb-rAb. Three nucleotides at the 5′ terminus of (N′)y andtwo nucleotides at the 3′ terminus of (N′)x may also comprise 3′-O-Mesugar modifications.

In one embodiment of Structure A1 or A2, five consecutive nucleotides atthe 5′ terminus of (N′)y or (N′)y-N2, respectively comprise the 2′-O-Mesugar modification and five consecutive nucleotides at the 3′ terminusof (N′)x comprise the 2′-O-Me sugar modification. In another embodimentten consecutive nucleotides at the 5′ terminus of (N′)y comprise the2′-O-Me sugar modification and five consecutive nucleotides at the 3′terminus of (N′)x comprise the 2′-O-Me sugar modification. In anotherembodiment thirteen consecutive nucleotides at the 5′ terminus of (N′)ycomprise the 2′-O-Me sugar modification; five consecutive nucleotides atthe 3′ terminus of (N′)x comprise the 2′-O-Me sugar modification; and Zand/or Z′ independently comprise one or more non-nucleotide moietyselected from the group consisting of an abasic moiety, an invertedabasic moiety, a hydrocarbon moiety, and an inorganic phosphate, or acombination of one or more non-nucleotide moiety and one or morenucleotide. In some embodiments Z is selected C3Pi-C3Ps; C3Pi-C3OH;C3Pi-C3Pi; C3Pi-rAb; C3Pi-dAb; dAb-dAb and rAb-rAb.

In specific embodiments five consecutive nucleotides at the 5′ terminusof (N′)y or (N′)y-N2, respectively comprise the 2′-O-Me sugarmodification and two consecutive nucleotides at the 3′ terminus of (N′)yare L-DNA. In addition, the compound may further comprise fiveconsecutive 2′-O-methyl modified nucleotides at the 3′ terminus of (N′)xand Z and/or Z′ may independently comprise one or more non-nucleotidemoiety selected from the group consisting of an abasic moiety, aninverted abasic moiety, a hydrocarbon moiety, and an inorganicphosphate, or a combination of one or more non-nucleotide moiety and oneor more nucleotide. In some embodiments Z is selected from C3Pi-C3Ps;C3Pi-C3OH; C3Pi-C3Pi; C3Pi-rAb; C3Pi-dAb; dAb-dAb and rAb-rAb.

In various embodiments of Structure A1 or A2 the modified nucleotides in(N)x are different from the modified nucleotides in (N′)y. For example,the modified nucleotides in (N)x are 2′ sugar modified nucleotides andthe modified nucleotides in (N′)y are nucleotides linked by 2′-5′internucleotide linkages. In another example, the modified nucleotidesin (N)x are mirror nucleotides and the modified nucleotides in (N′)y arenucleotides linked by 2′-5′ internucleotide linkages. In anotherexample, the modified nucleotides in (N)x are nucleotides linked by2′-5′ internucleotide linkages and the modified nucleotides in (N′)y aremirror nucleotides.

In some embodiments provided herein is a compound having a structure setforth below:

(X1) 5′(N)x-Z3′     (antisense strand) 3′Z′-(N′)y-z″5′(sense strand)wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or is an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein Z consists of two non-nucleotide moieties, or a combination of anon-nucleotide moiety and a nucleotide;wherein Z′ may be present or absent but if present comprises onenon-nucleotide moiety;wherein z″ may be present or absent but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;wherein each of x and y is independently an integer from 18 to 27;wherein (N′)y comprises at least one mirror nucleotide at the 3′terminus or 3′ penultimate position; andwherein the sequence of (N)x comprises an antisense sequence to amammalian gene.

In some embodiments x=y=19.

In some embodiments the mirror nucleotides is selected from an L-DNA andan L-RNA moiety. In some embodiments the mirror nucleotide is an L-DNAmoiety. In some embodiments either Z or Z′ is present and comprises anabasic moiety or hydrocarbon moiety or combination thereof. In someembodiments Z′ is absent, Z is present and comprises a hydrophobicmoiety.

In some embodiments the mirror nucleotides is selected from an L-DNA andan L-RNA moiety. In some embodiments the mirror nucleotide is an L-DNAmoiety.

In some embodiments N′(y) comprises two or 3 mirror nucleotides at the3′ terminus, and N(x) optionally comprises at least one mirrornucleotide at the 3′ terminus.

In some embodiments N′(y) comprises two mirror nucleotides at the 3′penultimate position. In some embodiments N(x) comprises one or twomirror nucleotides at the 3′ penultimate position and N′(y) optionallyfurther comprises one or two mirror nucleotides at the 5′ penultimateposition. In some embodiments (N′)y comprises one mirror nucleotide atthe 5′ terminus or 5′ penultimate position.

In some embodiments (N)x comprises 2′OMe sugar modified ribonucleotides.In some embodiments (N)x comprises 2′OMe sugar modified pyrimidineribonucleotides. In some embodiments (N)x comprises 2′OMe sugar modifiedribonucleotides alternating with unmodified ribonucleotides. In someembodiments x=y=19 and (N)x comprises 2′OMe sugar modifiedribonucleotides in position (5′>3′) 3, 5 and 11, 13, 15, 17, and 19. Insome embodiments (N)x further comprises a mirror nucleotide or a 2′5′nucleotide in positions 6 or 7.

In some embodiments the sequence of (N)x has complementarity to thesequence of (N′)y; and the sequence of (N′)y has identity to a sequencewithin an mRNA encoded by a target gene.

In another embodiment provided herein is a compound having the structureset forth below:

(X2) 5′(N)x-Z3′     (antisense strand) 3′Z′-(N′)y-z″5′(sense strand)wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or is an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein Z consists of two non-nucleotide moieties, or a combination of anon-nucleotide moiety and a nucleotide;wherein Z′ may be present or absent but if present comprises onenon-nucleotide moiety;wherein z″ may be present or absent but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;wherein each of x and y is independently an integer from 18 to 27;wherein (N′)y comprises at least one or more 2′OMe modified pyrimidinesandwherein the sequence of (N)x comprises an antisense sequence to amammalian gene.

In some embodiments x=y=19.

In some embodiments either Z or Z′ are present. In some embodiments bothZ and Z′ are present. In some embodiments Z′ is absent, Z is present andcomprises a hydrophobic moiety.

In some embodiments (N)x comprises 2′OMe sugar modified ribonucleotides.In some embodiments (N)x comprises 2′OMe sugar modified pyrimidineribonucleotides. In some embodiments (N)x comprises 2′OMe sugar modifiedribonucleotides alternating with unmodified ribonucleotides. In someembodiments x=y=19 and (N)x comprises 2′OMe sugar modifiedribonucleotides in position (5′>3′) 3, 5 and 11, 13, 15, 17, and 19. Insome embodiments (N)x further comprises a mirror nucleotide or a 2′5′nucleotide in positions 6 or 7.

In some embodiments the sequence of (N)x has complementarity to thesequence of (N′)y; and the sequence of (N′)y has identity to a sequencewithin an mRNA encoded by a target gene.

In some embodiments provide herein is a compound having a structure setforth below:

(X3) 5′(N)x-Z3′     (antisense strand) 3′Z′-(N′)y-z″5′(sense strand)wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein Z consists of two non-nucleotide moieties, or a combination of anon-nucleotide moiety and a nucleotide;wherein Z′ may be present or absent but if present comprises onenon-nucleotide moiety;wherein z″ may be present or absent but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;wherein each of x and y is independently an integer from 18 to 27;wherein (N′)y comprises at least one nucleotide joined to an adjacentnucleotide by a 2′-5′ internucleotide bond; andwherein the sequence of (N)x comprises an antisense sequence to amammalian gene.

In some embodiments x=y=19.

In some embodiments N′(y) comprises 2, 3, 4, 5, 6, 7, or 8 nucleotidesjoined to an adjacent nucleotide by a 2′-5′ internucleotide bond.

In some embodiments either Z or Z′ is present and comprises an abasicmoiety or hydrocarbon moiety or combination thereof. In some embodimentsZ′ is absent, Z is present and comprises a hydrophobic moiety.

In some embodiments N′(y) comprises 2, 3, 4, 5, 6, 7, or 8 nucleotidesjoined to an adjacent nucleotide by a 2′-5′ internucleotide bond at the3′ terminus. In some embodiments N′(y) comprises 2, 3, 4, 5, 6, 7, or 8nucleotides joined to an adjacent nucleotide by a 2′-5′ internucleotidebond at the 3′ penultimate position. In some embodiments x=y=19 andN′(y) comprises 2, 3, 4, or 5 nucleotides joined to an adjacentnucleotide by a 2′-5′ internucleotide bond at the 3′ terminus. In someembodiments x=y=19 and N′(y) comprises 5 nucleotides joined to anadjacent nucleotide by a 2′-5′ internucleotide bond at the 3′ terminusi.e. in position 15, 16, 17, 18 and 19 (5′>3′). In some embodiments (N)xcomprises 2′OMe sugar modified ribonucleotides. In some embodiments (N)xcomprises 2′OMe sugar modified pyrimidine ribonucleotides. In someembodiments (N)x comprises 2′OMe sugar modified ribonucleotidesalternating with unmodified ribonucleotides. In some embodiments x=y=19and (N)x comprises 2′OMe sugar modified ribonucleotides in position(5′>3′) 3, 5 and 11, 13, 15, 17, and 19. In some embodiments (N)xfurther comprises a mirror nucleotide or a 2′5′ nucleotide in positions6 or 7.

In some embodiments the sequence of (N)x has complementarity to thesequence of (N′)y; and the sequence of (N′)y has identity to a sequencewithin an mRNA encoded by a target gene.

In some preferred embodiment nucleic acid molecules disclosed hereinhave the following structure

5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′HOC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″ or5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′iPC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″wherein of N and N′ is independently a ribonucleotide which may beunmodified or modified, or is an unconventional moiety;wherein each N is linked to the adjacent N by a covalent bond;wherein each N′ is linked to the adjacent N′ by a covalent bond;wherein 1 to 10 of N are 2′-O Me sugar modified ribonucleotides;wherein N at position 5, 6, 7, 8 or 9 (5′>3′) is a 2′5 nucleotide or amirror nucleotide;wherein N′ at positions 15-19 (5′>3′) are 2′5′ ribonucleotides;wherein z″ is a capping moiety covalently attached to the 5′ terminus ofthe sense strand.

In some preferred embodiment nucleic acid molecules disclosed hereinhave the following structure

5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′HOC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″ or5′N N N N N N N N N N N N N N N N N N N-C3Pi-C3OH3′iPC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″wherein of N and N′ is independently a ribonucleotide which may beunmodified or modified, or is an unconventional moiety;wherein each N is linked to the adjacent N by a covalent bond;wherein each N′ is linked to the adjacent N′ by a covalent bond;wherein 1 to 10 of N are 2′-O Me sugar modified ribonucleotides;wherein N at position 5, 6, 7, 8 or 9 (5′>3′) is a 2′5 nucleotide or amirror nucleotide;wherein N′ comprises one or more 2′-O Me sugar modified pyrimidineribonucleotides;wherein N at position 9 or 10 (5′>3′) is a 2′5 nucleotide; andwherein z″ is a capping moiety covalently attached to the 5′ terminus ofthe sense strand.

In some embodiments of Structures (X1-X3), either the sense strand orthe antisense strand or both the sense and the antisense strandscomprise one or two inorganic phosphate moieties at the at the 3′termini.

In some embodiments of Structures (X1-X3) in (N)x the N at the 3′terminus is a modified ribonucleotide and (N)x comprises at least 8modified ribonucleotides. In some embodiments the modifiedribonucleotides comprise 2′ OMe sugar modified ribonucleotides. In otherembodiments at least 5 of the at least 8 modified ribonucleotides arealternating beginning at the 3′ end.

In various embodiments of Structures (X1-X3) z″ is present and isselected from an abasic ribose moiety, a deoxyribose moiety; an invertedabasic ribose moiety, a deoxyribose moiety; C6-amino-Pi; a mirrornucleotide.

In various embodiments of Structures (X1-X3) in (N′)y at least oneadditional unconventional moiety is present, which unconventional moietymay be an abasic ribose moiety, an abasic deoxyribose moiety, a modifiedor unmodified deoxyribonucleotide, a mirror nucleotide, a non-basepairing nucleotide analog or a nucleotide joined to an adjacentnucleotide by a 2′-5′ internucleotide phosphate bond. In someembodiments, neither (N)x nor (N′)y are phosphorylated at the 3′ and 5′termini. In other embodiments either or both (N)x and (N′)y arephosphorylated at the 3′ termini. In yet another embodiment, either orboth (N)x and (N′)y are phosphorylated at the 3′ termini usingnon-cleavable phosphate groups. In yet another embodiment, either orboth (N)x and (N′)y are phosphorylated at the terminal 2′ terminiposition using cleavable or non-cleavable phosphate groups.

In certain embodiments for all of the above-mentioned structures, Z ispresent. In other embodiments Z′ is present. In additional embodimentsboth Z and Z′ are present. In some embodiments Z and Z′ are both presentand identical. In further embodiments both Z and Z′ are present and aredifferent. In some embodiments Z and Z′ are independently 1, 2, 3, 4 or5 non-nucleotide moieties, or a combination of a non-nucleotide moietyand a nucleotide.

In some embodiments Z is present and comprises one or morenon-nucleotide moiety selected from an abasic moiety, an inverted abasicmoiety, a hydrocarbon moiety such as (CH2)3, and an inorganic phosphatemoiety.

In additional embodiments Z′ is present and comprises one or morenon-nucleotide moiety selected from an abasic moiety, an inverted abasicmoiety, a hydrocarbon moiety such as (CH2)3, and an inorganic phosphatemoiety.

In some embodiments each of Z and/or Z′ comprises one or twonon-nucleotide moieties and further comprises a nucleotide.

In some embodiments Z and/or Z′ comprise abasic moieties, optionallydeoxyribo-abasic (referred to herein as “dAb”) or riboabasic (referredto herein as “rAb”) moieties. In some embodiments each of Z and/or Z′ isdAb-dAb or rAb-rAb.

In some embodiments Z and/or Z′ comprise one or more hydrocarbonmoieties, optionally (CH2)3-Pi (referred to herein as “C3Pi”). In someembodiments Z and/or Z′ is C3Pi-C3Ps; C3Pi-C3OH; or C3Pi-C3Pi.

In additional embodiments Z and/or Z′ comprise a combination of abasicmoieties and unmodified nucleotides or a combination of hydrocarbonmodified moieties and unmodified nucleotides or a combination of abasicmoieties and hydrocarbon modified moieties. In such embodiments, Zand/or Z′ are optionally C3Pi-rAb. In a particular embodiment only Z ispresent and is C3Pi-C3Ps; C3Pi-C3OH; C3Pi-C3Pi.

In the embodiments of the above-mentioned Structures, the compoundcomprises at least one 3′ overhang (Z and or Z′) comprising at least onenon-nucleotide moiety. Z and Z′ independently comprises onenon-nucleotide moiety and one or more covalently linked modified ornon-modified nucleotides or unconventional moiety, for example inverteddT or dA; dT, LNA, mirror nucleotide and the like. The siRNA in which Zand/or Z′ is present has improved activity and/or stability and/oroff-target activity and or reduced immune response when compared to ansiRNA in which Z and/or Z′ are absent or in which Z and/or Z′ is dTdT.

In certain embodiments for all the above-mentioned Structures, thecompound comprises one or more phosphonocarboxylate and/orphosphinocarboxylate nucleotides (PACE nucleotides). In some embodimentsthe PACE nucleotides are deoxyribonucleotides and thephosphinocarboxylate nucleotides are phosphinoacetate nucleotides.Examples of PACE nucleotides and analogs are disclosed in U.S. Pat. Nos.6,693,187 and 7,067,641, both incorporated herein by reference.

In certain embodiments for all the above-mentioned Structures, thecompound comprises one or more locked nucleic acids (LNA) also definedas bridged nucleic acids or bicyclic nucleotides. Exemplary lockednucleic acids include 2′-O, 4′-C-ethylene nucleosides (ENA) or 2′-O,4′-C-methylene nucleosides. Other examples of LNA and ENA nucleotidesare disclosed in WO 98/39352, WO 00/47599 and WO 99/14226, allincorporated herein by reference.

In certain embodiments for all the above-mentioned Structures, thecompound comprises one or more altritol monomers (nucleotides), alsodefined as 1,5 anhydro-2-deoxy-D-altrito-hexitol (see for example,Allart, et al., 1998. Nucleosides & Nucleotides 17:1523-1526; Herdewijnet al., 1999. Nucleosides & Nucleotides 18:1371-1376; Fisher et al.,2007, NAR 35(4):1064-1074; all incorporated herein by reference).

The present invention explicitly excludes double stranded compounds inwhich each of N and/or N′ is a deoxyribonucleotide (dA, dC, dG, dT). Incertain embodiments (N)x and (N′)y may comprise independently 1, 2, 3,4, 5, 6, 7, 8, 9 or more deoxyribonucleotides. In certain embodimentsthe provided herein a compound wherein each of N is an unmodifiedribonucleotide and the 3′ terminal nucleotide or 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14 consecutive nucleotides at the 3′ terminus of (N′)yare deoxyribonucleotides. In yet other embodiments each of N is anunmodified ribonucleotide and the 5′ terminal nucleotide or 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive nucleotides at the 5′terminus of (N′)y are deoxyribonucleotides. In further embodiments the5′ terminal nucleotide or 2, 3, 4, 5, 6, 7, 8, or 9 consecutivenucleotides at the 5′ terminus and 1, 2, 3, 4, 5, or 6 consecutivenucleotides at the 3′ termini of (N)x are deoxyribonucleotides and eachof N′ is an unmodified ribonucleotide. In yet further embodiments (N)xcomprises unmodified ribonucleotides and 1 or 2, 3 or 4 consecutivedeoxyribonucleotides independently at each of the 5′ and 3′ termini and1 or 2, 3, 4, 5 or 6 consecutive deoxyribonucleotides in internalpositions; and each of N′ is an unmodified ribonucleotide. In certainembodiments the 3′ terminal nucleotide or 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12 13 or 14 consecutive nucleotides at the 3′ terminus of (N′)y andthe terminal 5′ nucleotide or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 13 or14 consecutive nucleotides at the 5′ terminus of (N)x aredeoxyribonucleotides. In some embodiments the 5′ terminal nucleotide ofN or 2 or 3 consecutive of N and 1,2, or 3 of N′ is adeoxyribonucleotide. Certain examples of active DNA/RNA siRNA chimerasare disclosed in US patent publication 2005/0004064, and Ui-Tei, 2008(NAR 36(7):2136-2151) incorporated herein by reference in theirentirety.

A covalent bond refers to an internucleotide linkage linking onenucleotide monomer to an adjacent nucleotide monomer. A covalent bondincludes for example, a phosphodiester bond, a phosphorothioate bond, aP-alkoxy bond, a P-carboxy bond and the like. The normal internucleosidelinkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. In certainembodiments a covalent bond is a phosphodiester bond. Covalent bondencompasses non-phosphorous-containing internucleoside linkages, such asthose disclosed in WO 2004/041924 inter alia. Unless otherwiseindicated, in embodiments of the structures discussed herein thecovalent bond between each consecutive N or N′ is a phosphodiester bond.

For all of the structures above, in some embodiments the oligonucleotidesequence of (N)x is fully complementary to the oligonucleotide sequenceof (N′)y. In other embodiments (N)x and (N′)y are substantiallycomplementary. In certain embodiments (N)x is fully complementary to atarget sequence. In other embodiments (N)x is substantiallycomplementary to a target sequence.

DEFINITIONS

For convenience certain terms employed in the specification, examplesand claims are described herein.

It is to be noted that, as used herein, the singular forms “a”, “an” and“the” include plural forms unless the content clearly dictatesotherwise.

Where aspects or embodiments of the invention are described in terms ofMarkush groups or other grouping of alternatives, those skilled in theart will recognize that the invention is also thereby described in termsof any individual member or subgroup of members of the group.

What is referred to herein as the “sense” or “sense strand” or“passenger strand” of a double stranded or duplex siRNA compound, refersto an oligonucleotide having identity to a target nucleic acid, forexample target RNA including target mRNA. What is referred to herein asthe “antisense” or “antisense strand” or “guide strand” refers to anoligonucleotide having complementarity to a target nucleic acid, forexample target mRNA. Without wishing to be bound to theory, theantisense, or guide strand, is incorporated into the RNA-inducedsilencing complex (RISC) and directs post-transcriptional genesilencing, which occurs when the guide strand base pairs with acomplementary sequence of a messenger RNA molecule and mediates cleavageof the mRNA by Argonaute, the catalytic component of RISC complex.

A “pro-apoptotic polypeptide” refers to a polypeptide encoded by any ofthe above listed genes, including splice variants, isoforms, orthologs,or paralogs and the like.

An “inhibitor” is a compound which is capable of reducing the expressionof a gene or the activity of the product of such gene to an extentsufficient to achieve a desired biological or physiological effect. Theterm “inhibitor” as used herein refers to one or more of anoligonucleotide inhibitor, including siRNA, shRNA, miRNA and ribozymesInhibition may also be referred to as down-regulation or, for RNAi,silencing.

The term “inhibit” as used herein refers to reducing the expression of agene or the activity of the product of such gene to an extent sufficientto achieve a desired biological or physiological effect. Inhibition maybe complete or partial.

As used herein, the terms “polynucleotide” and “nucleic acid” may beused interchangeably and refer to nucleotide sequences comprisingdeoxyribonucleic acid (DNA), and ribonucleic acid (RNA). The termsshould also be understood to include, as equivalents, analogs of eitherRNA or DNA made from nucleotide analogs. Throughout this applicationmRNA sequences are set forth as representing the target of theircorresponding genes. The terms “mRNA polynucleotide sequence” and mRNAare used interchangeably.

“Oligonucleotide” or “oligomer” refers to a deoxyribonucleotide orribonucleotide sequence from about 2 to about 50 nucleotides. Each DNAor RNA nucleotide may be independently natural or synthetic, and ormodified or unmodified. Modifications include changes to the sugarmoiety, the base moiety and or the linkages between nucleotides in theoligonucleotide. The compounds disclosed herein encompass moleculescomprising deoxyribonucleotides, ribonucleotides, modifieddeoxyribonucleotides, modified ribonucleotides and combinations thereof.As used herein, the terms “non-pairing nucleotide analog” means anucleotide analog which comprises a non-base pairing moiety includingbut not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole,3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-MedC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In someembodiments the non-base pairing nucleotide analog is a ribonucleotide.In other embodiments it is a deoxyribonucleotide.

Provided herein are methods and compositions for inhibiting expressionof a target gene in vivo. In general, the method includes administeringoligoribonucleotides, in particular small interfering RNAs (i.e.,siRNAs) or a nucleic acid material that generates siRNA in a cell, totarget a mammalian mRNA in an amount sufficient to down-regulateexpression of a target gene by an RNA interference mechanism. Inparticular, the method is useful for inhibiting expression of the genefor treatment of a subject suffering from a disease related toexpression of that gene. As disclosed herein the siRNA molecules orinhibitors of the target gene are used as drugs to treat variouspathologies.

“siRNA compound” and “nucleic acid molecule” may be used interchangeablyherein.

“Nucleotide” is meant to encompass a compound consisting of a nucleoside(a sugar, usually ribose or deoxyribose, and a purine or pyrimidinebase) and a phospho linker; such as a deoxyribonucleotide and aribonucleotide, which may be natural or synthetic, and be modified orunmodified. Modifications include changes and substitutions to the sugarmoiety, the base moiety and/or the internucleotide linkages.

A “phosphate based” moiety includes inorganic phosphate (Pi) andphosphorothioate (Ps).

All analogs of, or modifications to, a nucleotide/oligonucleotide may beemployed with the molecules disclosed herein, provided that said analogor modification does not substantially adversely affect the function ofthe nucleotide/oligonucleotide. Acceptable modifications includemodifications of the sugar moiety, modifications of the base moiety,modifications in the internucleotide linkages and combinations thereof.

What is sometimes referred to as an “abasic nucleotide” or “abasicnucleotide analog” is more properly referred to as a pseudo-nucleotideor an unconventional moiety. A nucleotide is a monomeric unit of nucleicacid, consisting of a ribose or deoxyribose sugar, a phosphate, and abase (adenine, guanine, thymine, or cytosine in DNA; adenine, guanine,uracil, or cytosine in RNA). A modified nucleotide comprises amodification in one or more of the sugar, phosphate and or base. Theabasic pseudo-nucleotide lacks a base, and thus is not strictly anucleotide. Abasic deoxyribose moiety includes for example abasicdeoxyribose-3′-phosphate; 1,2-dideoxy-D-ribofuranose-3-phosphate;1,4-anhydro-2-deoxy-D-ribitol-3-phosphate. Inverted abasic deoxyribosemoiety includes inverted deoxyriboabasic; 3′,5′ inverted deoxyabasic5′-phosphate. In general, an inverted abasic moiety is covalentlyattached to a 3′ terminal nucleotide via a 3′-3′ linkage; an invertedabasic moiety is covalently attached to a 5′ terminal nucleotide via a5′-5′ linkage; an inverted abasic moiety is generally covalentlyattached to an inverted abasic moiety via a 5′-3′ linkage.

The term “capping moiety” (z″) as used herein includes a moiety whichcan be covalently linked to the 5′ terminus of (N′)y and includes abasicribose moiety, abasic deoxyribose moiety, modified abasic ribose andabasic deoxyribose moieties including 2′ O alkyl modifications; invertedabasic ribose and abasic deoxyribose moieties and modifications thereof;C6-imino-Pi; a mirror nucleotide including L-DNA and L-RNA; 5′OMenucleotide; and nucleotide analogs including 4′,5′-methylene nucleotide;1-(β-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted abasic moiety; 1,4-butanediol phosphate;5′-amino; and bridging or non bridging methylphosphonate and 5′-mercaptomoieties.

Certain capping moieties are abasic ribose or abasic deoxyribosemoieties; inverted abasic ribose or abasic deoxyribose moieties;C6-amino-Pi; a mirror nucleotide including L-DNA and L-RNA. Thecompounds of the present invention may be synthesized using one or moreinverted nucleotides, for example inverted thymidine or inverted adenine(for example see Takei, et al., 2002. JBC 277(26):23800-06.

The term “non-nucleotide moiety” refers to a moiety that is not anucleotide, i.e. does not include all of the components of a nucleotide:a sugar. a base and a linker.

The term “unconventional moiety” as used herein refers to thenon-nucleotide moieties including an abasic moiety, an inverted abasicmoiety, a hydrocarbon (alkyl) moiety, and an inorganic phosphate andfurther includes a deoxyribonucleotide, a modified deoxyribonucleotide,a mirror nucleotide (L-DNA or L-RNA), a non-base pairing nucleotideanalog and a nucleotide joined to an adjacent nucleotide by a 2′-5′internucleotide phosphate bond (also known as 2′5′ nucleotide); bridgednucleic acids including LNA and ethylene bridged nucleic acids, linkagemodified (e.g. PACE) and base modified nucleotides as well as additionalmoieties explicitly disclosed herein as unconventional moieties.

When used in reference to the overhangs, an “alkyl moiety” or a“hydrocarbon moiety” refers to a C2, C3, C4, C5 or C6 straight chain orbranched alkyl moiety, including for example C2 (ethyl), C3 (propyl).When used in reference to the overhangs, a “derivative” of an alkyl or ahydrocarbon moiety refers to a C2, C3, C4, C5 or C6 straight chain orbranched alkyl moiety comprising a functional group which may beselected from among, inter alia, alcohols, phosphodiester,phosphorothioate, phosphonoacetate, amines, carboxylic acids, esters,amides and aldehydes.

When used in reference to modification of the ribose or deoxyribosemoiety, “alkyl” is intended to include linear, branched, or cyclicsaturated hydrocarbon structures and combinations thereof. “Loweralkyl”, when used in reference to modification of the ribose ordeoxyribose moiety, refers specifically to alkyl groups of from 1 to 6carbon atoms. Examples of lower alkyl groups include methyl, ethyl,propyl, isopropyl, butyl, s- and t-butyl and the like. Preferred alkylgroups are those of C₂₀ or below. Cycloalkyl is a subset of alkyl andincludes cyclic saturated hydrocarbon groups of from 3 to 8 carbonatoms. Examples of cycloalkyl groups include c-propyl, c-butyl,c-pentyl, norbornyl, adamantyl and the like

“Terminal functional group” includes halogen, alcohol, amine,carboxylic, ester, amide, aldehyde, ketone, ether groups.

In the context of the present invention, a “mirror” nucleotide (alsoreferred to as a spiegelmer) is a nucleotide analog with reversechirality to the naturally occurring or commonly employed nucleotide,i.e., a mirror image of the naturally occurring or commonly employednucleotide. The mirror nucleotide is a ribonucleotide (L-RNA) or adeoxyribonucleotide (L-DNA) and may further comprise at least one sugaror base modification and/or a backbone modification, such as aphosphorothioate or phosphonate moiety. U.S. Pat. No. 6,602,858discloses nucleic acid catalysts comprising at least one L-nucleotidesubstitution. Mirror nucleotide includes for example L-DNA(L-deoxyriboadenosine-3′-phosphate (mirror dA);L-deoxyribocytidine-3′-phosphate (mirror dC);L-deoxyriboguanosine-3′-phosphate (mirror dG);L-deoxyribothymidine-3′-phosphate (mirror image dT)) and L-RNA(L-riboadenosine-3′-phosphate (mirror rA); L-ribocytidine-3′-phosphate(mirror rC); L-riboguanosine-3′-phosphate (mirror rG);L-ribouracil-3′-phosphate (mirror dU).

Modified deoxyribonucleotide includes, for example 5′ OMe DNA(5-methyl-deoxyriboguanosine-3′-phosphate) which may be useful as anucleotide in the 5′ terminal position (position number 1); PACE(deoxyriboadenine 3′ phosphonoacetate, deoxyribocytidine 3′phosphonoacetate, deoxyriboguanosine 3′ phosphonoacetate,deoxyribothymidine 3′ phosphonoacetate.

Unconventional moieties include bridged nucleic acids including LNA(2′-0,4′-C-methylene bridged Nucleic Acid adenosine 3′ monophosphate,2′-0,4′-C-methylene bridged Nucleic Acid 5-methyl-cytidine 3′monophosphate, 2′-0,4′-C-methylene bridged Nucleic Acid guanosine 3′monophosphate, 5-methyl-uridine (or thymidine) 3′ monophosphate); andENA (2′-0,4′-C-ethylene bridged Nucleic Acid adenosine 3′ monophosphate,2′-0,4′-C-ethylene bridged Nucleic Acid 5-methyl-cytidine 3′monophosphate, 2′-0,4′-C-ethylene bridged Nucleic Acid guanosine 3′monophosphate, 5-methyl-uridine (or thymidine) 3′ monophosphate).

In some embodiments of the invention the unconventional moiety is anabasic ribose moiety, an abasic deoxyribose moiety, adeoxyribonucleotide, a mirror nucleotide, and a nucleotide joined to anadjacent nucleotide by a 2′-5′ internucleotide phosphate bond.

The nucleotides are selected from naturally occurring or syntheticmodified bases. Naturally occurring bases include adenine, guanine,cytosine, thymine and uracil. Modified bases of nucleotides includeinosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl andother alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine andother substituted guanines, other aza and deaza adenines, other aza anddeaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine. siRNAcompounds comprising one or more abasic pseudo-nucleotides areencompassed by the present invention. A nucleotide monomer comprising amodified base, including abasic pseudo-nucleotide monomers, may besubstituted for one or more ribonucleotides of the oligonucleotide. Anabasic pseudo-nucleotide monomer may be included at the one or more ofthe terminal positions or as a 5′ terminal cap. A 5′ terminal cap mayalso be selected from an inverted abasic pseudo-nucleotide analog, anL-DNA nucleotide, and a C6-imine phosphate.

In addition, analogues of polynucleotides are prepared wherein thestructure of one or more nucleotide is fundamentally altered and bettersuited as therapeutic or experimental reagents. An example of anucleotide analog is a peptide nucleic acid (PNA) wherein thedeoxyribose (or ribose) phosphate backbone in DNA (or RNA comprises witha polyamide backbone which is similar to that found in peptides. PNAanalogs have been shown to be resistant to enzymatic degradation and tohave extended lives in vivo and in vitro.

Possible modifications to the sugar residue are manifold and include2′-O alkyl, 2′-halo (e.g. 2′ deoxy fluoro), locked nucleic acid (LNA),glycol nucleic acid (GNA), threose nucleic acid (TNA), arabinoside;altritol (ANA) and other 6-membered sugars including morpholinos, andcyclohexinyls. Possible modifications on the 2′ moiety of the sugarresidue include amino, fluoro, methoxy alkoxy, alkyl, amino, fluoro,chloro, bromo, CN, CF, imidazole, carboxylate, thioate, C₁ to C₁₀ loweralkyl, substituted lower alkyl, alkaryl or aralkyl, OCF₃, OCN, O-, S-,or N-alkyl; O-, S, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂, N₃;heterozycloalkyl; heterozycloalkaryl; aminoalkylamino; polyalkylamino orsubstituted silyl, as, among others, described in European patents EP 0586 520 B1 or EP 0 618 925 B1. One or more deoxyribonucleotides are alsotolerated in the compounds of the present invention. In some embodiments(N′) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 DNA moieties.

LNA compounds are disclosed in International Patent Publication Nos. WO00/47599, WO 99/14226, and WO 98/39352. Examples of siRNA compoundscomprising LNA nucleotides are disclosed in Elmen et al., (NAR 2005.33(1):439-447) and in International Patent Publication No. WO2004/083430. Six-membered ring nucleotide analogs are disclosed inAllart, et al (Nucleosides & Nucleotides, 1998, 17:1523-1526; andPerez-Perez, et al., 1996, Bioorg. and Medicinal Chem Letters6:1457-1460) Oligonucleotides comprising 6-membered ring nucleotideanalogs including hexitol and altritol nucleotide monomers are disclosedin International patent application publication No. WO 2006/047842.

Backbone modifications, also known as internucleotide linkagemodifications, such as ethyl (resulting in a phospho-ethyl triester);propyl (resulting in a phospho-propyl triester); and butyl (resulting ina phospho-butyl triester) are also possible. Other backbonemodifications include polymer backbones, cyclic backbones, acyclicbackbones, thiophosphate-D-ribose backbones, amidates, phosphonoacetatederivatives. Certain structures include siRNA compounds having one or aplurality of 2′-5′ internucleotide linkages (bridges or backbone).

In some embodiments, neither (N)x nor (N′)y are phosphorylated at the 3′and 5′ termini. In other embodiments either or both (N)x and (N′)y arephosphorylated at the 3′ termini (3′ Pi). In yet another embodiment,either or both (N)x and (N′)y are phosphorylated at the 3′ termini withnon-cleavable phosphate groups. In yet another embodiment, either orboth (N)x and (N′)y are phosphorylated at the terminal 2′ terminiposition using cleavable or non-cleavable phosphate groups. Further, theinhibitory nucleic acid molecules of the present invention may compriseone or more gaps and/or one or more nicks and/or one or more mismatches.Without wishing to be bound by theory, gaps, nicks and mismatches havethe advantage of partially destabilizing the nucleic acid/siRNA, so thatit may be more easily processed by endogenous cellular machinery such asDICER, DROSHA or RISC into its inhibitory components.

In the context of the present invention, a gap in a nucleic acid refersto the absence of one or more internal nucleotides in one strand, whilea nick in a nucleic acid refers to the absence of an internucleotidelinkage between two adjacent nucleotides in one strand. Any of themolecules of the present invention may contain one or more gaps and/orone or more nicks.

Oligonucleotides

In a non-limiting example, Tables B (B1-B74), Tables C (C1-C4) andTables D (D1-D34) of PCT Patent Application Publication No. WO2009/044392, assigned to the assignee of the present invention andincorporated by reference in its entirety, comprise nucleic acidsequences of sense and corresponding antisense oligomers, useful inpreparing siRNA compounds according to the present application. Thecompounds are used as chemically and or structurally modified compounds.

The selection and synthesis of siRNA corresponding to known genes hasbeen widely reported; see for example Ui-Tei et al., J BiomedBiotechnol. 2006; 65052; Chalk et al., BBRC. 2004, 319(1):264-74; Sioud& Leirdal, Met. Mol Biol. 2004, 252:457-69; Levenkova et al., Bioinform.2004, 20(3):430-2; Ui-Tei et al., NAR. 2004, 32(3):936-48. For examplesof the use and production of modified siRNA see for example Braasch etal., Biochem. 2003, 42(26):7967-75; Chiu et al., RNA. 2003,9(9):1034-48; PCT Publication Nos. WO 2004/015107 and WO 02/44321 andU.S. Pat. Nos. 5,898,031 and 6,107,094.

The present invention provides double-stranded oligonucleotides (e.g.siRNAs), which down-regulate the expression of a desired gene. A siRNAof the invention is a duplex oligoribonucleotide in which the sensestrand is derived from the mRNA sequence of the desired gene, and theantisense strand is at least substantially complementary to the sensestrand. In general, some deviation from the target mRNA sequence istolerated without compromising the siRNA activity (see e.g. Czauderna etal., NAR. 2003, 31(11):2705-2716). An siRNA of the invention inhibitsgene expression on a post-transcriptional level with or withoutdestroying the mRNA. Without being bound by theory, siRNA may target themRNA for specific cleavage and degradation and/or may inhibittranslation from the targeted message.

In other embodiments at least one of the two strands may have anoverhang of at least one nucleotide at the 5′-terminus; the overhang mayconsist of at least one deoxyribonucleotide. The length of RNA duplex isfrom about 16 to about 40 ribonucleotides, preferably 19ribonucleotides. Further, the length of each strand may independentlyhave a length selected from the group consisting of about 16 to about 40bases, preferably 18 to 23 bases and more preferably 19 ribonucleotides.

In certain embodiments the complementarity between said first strand andthe target nucleic acid is perfect. In some embodiments, the strands aresubstantially complementary, i.e. having one, two or up to fivemismatches between said first strand and the target mRNA or between thefirst and the second strands. Substantially complementary refers tocomplementarity of greater than about 70%, and less than 100% to anothersequence. For example in a duplex region consisting of 19 base pairs onemismatch results in 94.7% complementarity, two mismatches results inabout 89.5% complementarity, 3 mismatches results in about 84.2%complementarity, 4 mismatches results in about 79% complementarity and 5mismatches results in about 74% complementarity, rendering the duplexregion substantially complementary. Accordingly, substantially identicalrefers to identity of greater than about 70%, to another sequence.

The first strand and the second strand may be linked by a loopstructure, which may be comprised of a non-nucleic acid polymer such as,inter alia, polyethylene glycol. Alternatively, the loop structure maybe comprised of a nucleic acid, including modified and non-modifiedribonucleotides and modified and non-modified deoxyribonucleotides.

Further, the 5′-terminus of the first strand of the siRNA may be linkedto the 3′-terminus of the second strand, or the 3′-terminus of the firststrand may be linked to the 5′-terminus of the second strand, saidlinkage being via a nucleic acid linker typically having a lengthbetween 2-100 nucleobases, preferably about 2 to about 30 nucleobases.

In some embodiments of the compounds of the invention having alternatingribonucleotides modified in at least one of the antisense and the sensestrands of the compound, for 19 mer and 23 mer oligomers theribonucleotides at the 5′ and 3′ termini of the antisense strand aremodified in their sugar residues, and the ribonucleotides at the 5′ and3′ termini of the sense strand are unmodified in their sugar residues.For 21 mer oligomers the ribonucleotides at the 5′ and 3′ termini of thesense strand are modified in their sugar residues, and theribonucleotides at the 5′ and 3′ termini of the antisense strand areunmodified in their sugar residues, or may have an optional additionalmodification at the 3′ terminus. As mentioned above, in some embodimentsthe middle nucleotide of the antisense strand is unmodified.

According to one embodiment of the invention, the antisense and thesense strands of the oligonucleotide/siRNA are phosphorylated only atthe 3′-terminus and not at the 5′-terminus. According to anotherembodiment of the invention, the antisense and the sense strands arenon-phosphorylated. According to yet another embodiment of theinvention, the 5′ most ribonucleotide in the sense strand is modified toabolish any possibility of in vivo 5′-phosphorylation.

Any siRNA sequence disclosed herein are prepared having any of themodifications/structures disclosed herein. The combination of sequenceplus structure is novel and is useful used in the treatment of theconditions disclosed herein.

Pharmaceutical Compositions

While it may be possible for the compounds of the present invention tobe administered as the raw chemical, it is preferable to present them asa pharmaceutical composition. Accordingly the present invention providesa pharmaceutical composition comprising one or more of the compounds ofthe invention; and a pharmaceutically acceptable carrier. Thiscomposition may comprise a mixture of two or more differentoligonucleotides/siRNAs.

The invention further provides a pharmaceutical composition comprisingat least one compound of the invention covalently or non-covalentlybound to one or more compounds of the invention in an amount effectiveto inhibit one or more genes as disclosed above; and a pharmaceuticallyacceptable carrier. The compound may be processed intracellularly byendogenous cellular complexes to produce one or moreoligoribonucleotides of the invention.

The invention further provides a pharmaceutical composition comprising apharmaceutically acceptable carrier and one or more of the compounds ofthe invention in an amount effective to down-regulate expression in acell of a target RNA, including target genes and target mRNA, and ortarget protein, the compound comprising a sequence havingcomplementarity to the sequence of (N)_(x). In certain embodiments, thetarget gene is a viral, bacterial or mammalian gene. In variousembodiments the target gene is a mammalian gene, preferably a humangene.

Additionally, the invention provides a method of inhibiting theexpression of a target gene, by at least 50% as compared to a control,comprising contacting an mRNA transcript of the target gene with one ormore of the compounds of the invention. In some embodiments an activesiRNA compound inhibits gene expression at a level of at least 50%, 60%or 70% as compared to control. In certain embodiments inhibition is at alevel of at least 75%, 80% or 90% as compared to control. In someembodiments the target gene is a pro-apoptotic gene as disclosed herein.

In one embodiment the oligoribonucleotide is inhibiting one or more ofthe pro-apoptotic genes of the present invention, whereby the inhibitionis selected from the group comprising inhibition of gene function,inhibition of polypeptide and inhibition of mRNA expression.

In one embodiment the compound inhibits expression of a polypeptideencoded by a target gene whereby the inhibition is selected from thegroup comprising inhibition of function (which may be examined by anenzymatic assay or a binding assay with a known interactor of the nativegene/polypeptide, inter alia), inhibition of protein (which may beexamined by Western blotting, ELISA or immuno-precipitation, inter alia)and inhibition of mRNA expression (which may be examined by Northernblotting, quantitative RT-PCR, in-situ hybridisation or microarrayhybridisation, inter alia).

In additional embodiments the invention provides a method of treating asubject suffering from a disease accompanied by an elevated level of thepro-apoptotic genes of the present invention, the method comprisingadministering to the subject a compound of the invention in atherapeutically effective dose thereby treating the subject.

Delivery

In some embodiments the siRNA molecules of the present invention aredelivered to the target tissue by direct application of the nakedmolecules prepared with a carrier or a diluent.

The term “naked siRNA” refers to siRNA molecules that are free from anydelivery vehicle that acts to assist, promote or facilitate entry intothe cell, including viral sequences, viral particles, liposomeformulations, lipofectin or precipitating agents and the like. Forexample, siRNA in PBS is “naked siRNA”.

However, in some embodiments the siRNA molecules of the invention aredelivered in liposome or lipofectin formulations and the like and areprepared by methods well known to those skilled in the art. Such methodsare described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference.

Delivery systems aimed specifically at the enhanced and improveddelivery of siRNA into mammalian cells have been developed, (see, forexample, Shen et al FEBS Let. 2003, 539:111-114; Xia et al., Nat.Biotech. 2002, 20:1006-1010; Reich et al., Mol. Vision 2003, 9: 210-216;Sorensen et al., J. Mol. Biol. 2003. 327: 761-766; Lewis et al., Nat.Gen. 2002, 32: 107-108 and Simeoni et al., NAR 2003, 31,11: 2717-2724).siRNA has recently been successfully used for inhibition of geneexpression in primates (see for example, Tolentino et al., Retina24(4):660.

The pharmaceutically acceptable carriers, solvents, diluents,excipients, adjuvants and vehicles as well as implant carriers generallyrefer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention and they include liposomes and microspheres. Examples ofdelivery systems useful in the present invention include U.S. Pat. Nos.5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603;4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many othersuch implants, delivery systems, and modules are well known to thoseskilled in the art. In one specific embodiment of this invention topicaland transdermal formulations may be selected. The siRNAs orpharmaceutical compositions of the present invention are administeredand dosed in accordance with good medical practice, taking into accountthe clinical condition of the individual patient, the disease to betreated, the site and method of administration, scheduling ofadministration, patient age, sex, body weight and other factors known tomedical practitioners.

The “therapeutically effective dose” for purposes herein is thusdetermined by such considerations as are known in the art. The dose mustbe effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In general, Dosage may be from 0.01 mg to 1 g per kg of body weight(e.g., 0.1 mg, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 25mg, 50 mg, 100 mg, 250 mg, 500 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50mg, 100 mg, 250 mg, or 500 mg per kg).

A suitable dosage unit of nucleic acid molecules may be in the range of0.001 to 0.25 milligrams per kilogram body weight of the recipient perday, or in the range of 0.01 to 20 micrograms per kilogram body weightper day, or in the range of 0.01 to 10 micrograms per kilogram bodyweight per day, or in the range of 0.10 to 5 micrograms per kilogrambody weight per day, or in the range of 0.1 to 2.5 micrograms perkilogram body weight per day.

Suitable amounts of nucleic acid molecules may be administered and theseamounts can be empirically determined using standard methods. Effectiveconcentrations of individual nucleic acid molecule species in theenvironment of a cell may be about 1 femtomolar (fmolar), about 50femtomolar, 100 femtomolar, 1 picomolar, 1.5 picomolar, 2.5 picomolar, 5picomolar, 10 picomolar, 25 picomolar, 50 picomolar, 100 picomolar, 500picomolar, 1 nanomolar, 2.5 nanomolar, 5 nanomolar, 10 nanomolar, 25nanomolar, 50 nanomolar, 100 nanomolar, 500 nanomolar, 1 micromolar, 2.5micromolar, 5 micromolar, 10 micromolar, 100 micromolar or more.

The amount of active ingredient that can be combined with the carriermaterials to produce a single dosage form varies depending upon the hosttreated and the particular mode of administration. Dosage unit formsgenerally contain between from about 1 mg to about 500 mg of an activeingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

Pharmaceutical compositions that include the nucleic acid moleculedisclosed herein may be administered once daily, qid, tid, bid, QD, orat any interval and for any duration that is medically appropriate.However, the therapeutic agent may also be dosed in dosage unitscontaining two, three, four, five, six or more sub-doses administered atappropriate intervals throughout the day. In that case, the nucleic acidmolecules contained in each sub-dose may be correspondingly smaller inorder to achieve the total daily dosage unit. The dosage unit can alsobe compounded for a single dose over several days, e.g., using aconventional sustained release formulation which provides sustained andconsistent release of the dsRNA over a several day period. Sustainedrelease formulations are well known in the art. The dosage unit maycontain a corresponding multiple of the daily dose. The composition canbe compounded in such a way that the sum of the multiple units of anucleic acid together contain a sufficient dose.

Kits and Containers

Also provided are kits, containers and formulations that include anucleic acid molecule (e.g., an siNA molecule) as provided herein forreducing expression of a target gene for administering the nucleic acidmolecule to a subject. In some embodiments a kit includes at least onecontainer and at least one label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers can beformed from a variety of materials such as glass, metal or plastic. Kitsmay further include associated indications and/or directions; reagentsand other compositions or tools used for such purpose can also beincluded.

The container can alternatively hold a composition that is effective fortreating, diagnosis, prognosing or prophylaxing a condition and can havea sterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agents in the composition can be a nucleicacid molecule capable of specifically binding a target gene and/ormodulating the function of a target gene.

A kit may further include a second container that includes apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and/or dextrose solution. It can further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, stirrers, needles, syringes, and/orpackage inserts with indications and/or instructions for use.

The units dosage ampoules or multidose containers, in which the nucleicacid molecules are packaged prior to use, may include an hermeticallysealed container enclosing an amount of nucleic acid molecules orsolution containing nucleic acid molecules suitable for apharmaceutically effective dose thereof, or multiples of an effectivedose. The nucleic acid molecules are packaged as a sterile formulation,and the hermetically sealed container is designed to preserve sterilityof the formulation until use.

The container comprising the nucleic acid molecules may include apackage that is labeled, and the label may bear a notice in the formprescribed by a governmental agency, for example the Food and DrugAdministration, which notice is reflective of approval by the agencyunder Federal law, of the manufacture, use, or sale of thepolynucleotide material therein for human administration.

Federal or National law requires that the use of pharmaceuticalcompositions in the therapy of humans be approved by an agency of theFederal or National government. In the United States, enforcement is theresponsibility of the Food and Drug Administration, which issuesregulations for securing such approval, detailed in 21 U.S.C. §301-392.Similar approval is required by most foreign countries and uniqueprocedures are well known to those in the art and the compositions andmethods provided herein preferably comply accordingly.

The dosage to be administered depends to a large extent on the conditionand size of the subject being treated as well as the frequency oftreatment and the route of administration. Regimens for continuingtherapy, including dose and frequency may be guided by the initialresponse and clinical judgment.

The nucleic acid compounds disclosed herein are administered by any ofthe conventional routes of administration. It should be noted that thecompound is administered as the compound, per se, or as pharmaceuticallyacceptable salt and is administered alone or as an active ingredient incombination with pharmaceutically acceptable carriers, solvents,diluents, excipients, adjuvants and vehicles. The compounds areadministered orally, topically, subcutaneously or parenterally includingintravenous, intraarterial, intramuscular, intraperitoneally, andintranasal, inhalation, transtympanic administration as well asintrathecal and infusion techniques. Implants of the compounds are alsouseful. Liquid forms may be prepared for injection, the term includingsubcutaneous, transdermal, intravenous, intramuscular, intrathecal, andother parental routes of administration. The liquid compositions includeaqueous solutions, with and without organic co-solvents, aqueous or oilsuspensions, emulsions with edible oils, as well as similarpharmaceutical vehicles. In a particular embodiment, the administrationcomprises intravenous administration. In another embodiment theadministration comprises topical or local administration. In someembodiments topical administration includes topical administration tothe mammalian ear canal. In some embodiments topical administrationincludes topical administration to the surface of a mammalian eye.

In addition, in certain embodiments the compositions for use in thenovel treatments of the present invention may be formed as aerosols, forexample for intranasal administration.

In certain embodiments, oral compositions (such as tablets, suspensions,solutions) may be effective for local delivery to the oral cavity suchas oral composition suitable for mouthwash for the treatment of oralmucositis.

In embodiments the subject being treated is a warm-blooded animal and,in particular, mammals including human.

In an additional embodiment, the modification is a modification of thephosphate moiety, whereby the modified phosphate moiety is selected fromthe group comprising phosphorothioate or lack of a phosphate group.

The molecules of the present invention comprise siRNA, siNA, syntheticsiRNAs, synthetic shRNAs, and miRNA in addition to other nucleic acidsequences or molecules which encode such molecules or other inhibitorynucleotide molecules.

In some embodiments the nucleic acid compounds are useful for diagnosis.Without wishing to be bound to theory a double stranded nucleic acidmolecule comprising a 3′ terminal non-nucleotide can be efficientlydelivered to specific cells and tissue and are useful in diagnosis ofdisorders on the specific cell or tissue. According, end modificationsinclude detectable moieties including colorgenic agents, radiolabeledmoieties and enymatic agents. In some embodiments the detectable agentis a biotin group. Such biotin group may preferably be attached toeither the most 5′ nucleotide of the sense strand or the most 3′nucleotide of the antisense strand or to both of those ends. The variousend modifications as disclosed herein are preferably located at theribose moiety of a nucleotide of the nucleic acid according to thepresent invention. More particularly, the end modification may beattached to or replace any of the OH-groups of the ribose moiety,including but not limited to the 2′OH, 3′OH and 5′OH position, providedthat the nucleotide thus modified is a terminal nucleotide, preferably a5′ terminal nucleotide of the sense strand. It is to be understood thatthe nucleic acid molecules disclosed herein, or any long double-strandedRNA molecules (typically 25-500 nucleotides in length) which areprocessed by endogenous cellular complexes (such as DICER—see above) toform the siRNA molecules disclosed herein, or molecules which comprisethe siRNA molecules disclosed herein, are incorporated into themolecules of the present invention to form additional novel molecules,and are employed in the treatment of the diseases or disorders describedherein.

In particular, it is envisaged that a long oligonucleotide may bedelivered in a carrier, preferably a pharmaceutically acceptablecarrier, and may be processed intracellularly by endogenous cellularcomplexes (e.g. by DROSHA and DICER as described above) to produce oneor more smaller double stranded oligonucleotides (siRNAs) which areoligonucleotides of the invention. This oligonucleotide is termed atandem shRNA construct. It is envisaged that this long oligonucleotideis a single stranded oligonucleotide comprising one or more stem andloop structures, wherein each stem region comprises a sense andcorresponding antisense siRNA sequence. Any molecules, such as, forexample, antisense DNA molecules which comprise the inhibitory sequencesdisclosed herein (with the appropriate nucleic acid modifications) areparticularly desirable and may be used in the same capacity as theircorresponding RNAs/siRNAs for all uses and methods disclosed herein.

Backbone

The nucleoside subunits of the nucleic acid molecules disclosed hereinmay be linked to each other by phosphodiester bonds. The standard 5′3′phosphodiester bond may be optionally substituted with other linkages.For example, phosphorothioate, thiophosphate-D-ribose entities,triester, thioate, 2′-5′ bridged backbone (may also be referred to as5′-2′ or 2′5′), PACE, 3′-(or -5′)deoxy-3′-(or -5′)thio-phosphorothioate,phosphorodithioate, phosphoroselenates, 3′-(or -5′)deoxy phosphinates,borano phosphates, 3′-(or -5′)deoxy-3′-(or 5′-)amino phosphoramidates,hydrogen phosphonates, phosphonates, borano phosphate esters, alkyl oraryl phosphonates and phosphotriester modifications such asalkylphosphotriesters, phosphotriester phosphorus linkages,5′-ethoxyphosphodiester, P-alkyloxyphosphotriester, methylphosphonate,and nonphosphorus containing linkages for example, carbonate, carbamate,silyl, sulfur, sulfonate, sulfonamide, formacetal, thioformacetyl,oxime, methyleneimino, methylenemethylimino, methylenehydrazo,methylenedimethylhydrazo and methyleneoxymethylimino linkages. Inaddition, analogs of polynucleotides can be prepared wherein thestructure of the nucleotide is fundamentally altered and that are bettersuited as therapeutic or experimental reagents. An example of anucleotide analog is a peptide nucleic acid (PNA) wherein thedeoxyribose (or ribose) phosphate backbone in DNA (or RNA comprises apolyamide backbone which is similar to that found in peptides. PNAanalogs have been shown to be resistant to degradation by enzymes and tohave extended lives in vivo and in vitro. Further, PNAs have been shownto bind stronger to a complementary DNA sequence than a DNA molecule.This observation is attributed to the lack of charge repulsion betweenthe PNA strand and the DNA strand. Other modified monomers useful insynthesizing the oligonucleotides include moieties having polymerbackbones, cyclic backbones, or acyclic backbones.

Methods of Treatment

In another aspect, the present invention relates to a method for thetreatment of a subject in need of treatment for a disease or disorderassociated with the abnormal expression of a target gene, comprisingadministering to the subject an amount of an inhibitor which reduces orinhibits expression of the gene.

In certain embodiments the subject being treated is a warm-bloodedanimal and, in particular, mammals including human.

The methods of the invention comprise administering to the subject oneor more inhibitory compounds which down-regulate expression of a gene;and in particular siRNA in a therapeutically effective dose so as tothereby treat the subject.

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) a disorder as listed herein. Those in need oftreatment include those already experiencing the disease or condition,those prone to having the disease or condition, and those in which thedisease or condition is to be prevented. The compounds of the inventionare administered before, during or subsequent to the onset of thedisease or condition or symptoms associated therewith. In cases wheretreatment is for the purpose of prevention, then the present inventionrelates to a method for delaying the onset of or averting thedevelopment of the disease or disorder.

The present invention relates to the use of compounds whichdown-regulate the expression of the pro-apoptotic genes of the inventionparticularly to novel small interfering RNAs (siRNAs), in the treatmentof the following diseases or conditions in which inhibition of theexpression of the pro-apoptotic genes is beneficial: hearing loss, acuterenal failure (ARF), glaucoma, acute respiratory distress syndrome(ARDS) and other acute lung and respiratory injuries,ischemia-reperfusion injury following lung transplantation, organtransplantation including lung, liver, heart, bone marrow, pancreas,cornea and kidney transplantation which includes DGF; spinal cordinjury, pressure sores, age-related macular degeneration (AMD), dry eyesyndrome, ocular ischemic conditions including ION and NAION; oralmucositis and chronic obstructive pulmonary disease (COPD). Otherindications include chemical-induced nephrotoxicity and chemical-inducedneurotoxicity, for example toxicity induced by cisplatin andcisplatin-like compounds, by aminoglycosides, by loop diuretics, and byhydroquinone and their analogs.

Methods, molecules and compositions which inhibit the pro-apoptoticgenes of the invention are discussed herein at length, and any of saidmolecules and/or compositions may be beneficially employed in thetreatment of a subject suffering from any of said conditions.

In a further aspect an article of manufacture is provided which includespackaging material comprising an oligonucleotide composition accordingto the invention that is therapeutically effective in treating a subjectsuffering from any one of the indications disclosed herein, andinstructions for use.

Disclosed herein is a method of preparing a double-stranded RNA moleculecapable of target-specific inhibition or down-regulating expression of atarget gene wherein each RNA strand has a length from 19 to 25nucleotides, wherein at least one strand has a non-nucleotide moietycovalently attached at a 3′ or a 2′ position of the sugar residue at the3′ terminal end thereof, comprising (a) synthesizing two RNA strandseach having a length from 19 to 25 nucleotides, wherein said RNA strandsare capable of forming a double-stranded RNA molecule, (b) combining thesynthesized RNA strands under conditions, wherein a double-stranded RNAmolecule is formed, wherein said double-stranded RNA molecule consistsof a single double stranded region and at least one single strandedregion comprising a non-nucleotide moiety covalently attached at a 3′ ora 2′ position of the sugar residue at the 3′ terminal end of the strandin which it is present; wherein the non-nucleotide moiety is selectedfrom propanol, a C3 alkyl moiety linked to a phosphodiester, a C3 alkylmoiety linked to a phosphorothioate, a deoxyriboabasic moiety or ariboabasic moiety and a combination thereof.

Provided herein is a process of preparing a pharmaceutical composition,which comprises:

-   -   providing one or more compounds disclosed herein; and    -   admixing said compound with a pharmaceutically acceptable        carrier.

The present invention also provides for a process of preparing apharmaceutical composition, which comprises admixing one or morecompounds of the present invention with a pharmaceutically acceptablecarrier.

In one embodiment, the compound used in the preparation of apharmaceutical composition is admixed with a carrier in apharmaceutically effective dose. In a particular embodiment the compoundof the present invention is conjugated to a steroid or to a lipid or toanother suitable molecule e.g. to cholesterol.

Synthesis of Modified Compounds

The compounds of the present invention can be synthesized by any of themethods that are well known in the art for synthesis of ribonucleic (ordeoxyribonucleic) oligonucleotides. Such synthesis is, among others,described in Beaucage and Iyer, Tetrahedron 1992; 48:2223-2311; Beaucageand Iyer, Tetrahedron 1993; 49: 6123-6194 and Caruthers, et. al.,Methods Enzymol. 1987; 154: 287-313; the synthesis of thioates is, amongothers, described in Eckstein, Annu. Rev. Biochem. 1985; 54: 367-402,the synthesis of RNA molecules is described in Sproat, in Humana Press2005 edited by Herdewijn P.; Kap. 2: 17-31 and respective downstreamprocesses are, among others, described in Pingoud et. al., in IRL Press1989 edited by Oliver R. W. A.; Kap. 7: 183-208.

Other synthetic procedures are known in the art e.g. the procedures asdescribed in Usman et al., J. Am. Chem. Soc., 1987, 109:7845; Scaringeet al., NAR, 1990, 18:5433; Wincott et al., NAR 1995. 23:2677-2684; andWincott et al., Methods Mol. Bio., 1997, 74:59, and these procedures maymake use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. Themodified (e.g. 2′-O-methylated) nucleotides and unmodified nucleotidesare incorporated as desired.

The oligonucleotides of the present invention can be synthesizedseparately and joined together post-synthetically, for example, byligation (Moore et al., Science 1992, 256:9923; International PatentPublication No. WO 93/23569; Shabarova et al., NAR 1991, 19:4247; Bellonet al., Nucleosides & Nucleotides, 1997, 16:951; Bellon et al.,Bioconjugate Chem 1997, 8:204), or by hybridization following synthesisand/or deprotection.

It is noted that a commercially available machine (available, interalia, from Applied Biosystems) can be used; the oligonucleotides areprepared according to the sequences disclosed herein. Overlapping pairsof chemically synthesized fragments can be ligated using methods wellknown in the art (e.g., see U.S. Pat. No. 6,121,426). The strands aresynthesized separately and then are annealed to each other in the tube.Then, the double-stranded siRNAs are separated from the single-strandedoligonucleotides that were not annealed (e.g. because of the excess ofone of them) by HPLC. In relation to the siRNAs or siRNA fragments ofthe present invention, two or more such sequences can be synthesized andlinked together for use in the present invention.

The compounds of the invention can also be synthesized via tandemsynthesis methodology, as described for example in US Patent PublicationNo. 2004/0019001 (McSwiggen), wherein both siRNA strands are synthesizedas a single contiguous oligonucleotide fragment or strand separated by acleavable linker which is subsequently cleaved to provide separate siRNAfragments or strands that hybridize and permit purification of the siRNAduplex. The linker is selected from a polynucleotide linker or anon-nucleotide linker.

The term “Covalent bonding” as used herein refers to chemical bondingthat is characterized by the sharing of pairs of electrons betweenatoms.

The term “Noncovalent bonding” as used herein refers to a variety ofinteractions that are not covalent in nature between molecules or partsof molecules that provide force to hold the molecules or parts ofmolecules together, usually in a specific orientation or conformation.These noncovalent interactions include: ionic bonds, hydrophobicinteractions, hydrogen bonds, Van der Waals forces and dipole-dipolebonds.

siRNAs and RNA Interference

RNA interference (RNAi) is a phenomenon involving double-stranded (ds)RNA-dependent gene specific posttranscriptional silencing. Initialattempts to study this phenomenon and to manipulate mammalian cellsexperimentally were frustrated by an active, non-specific antiviraldefense mechanism which was activated in response to long dsRNAmolecules (Gil et al. Apoptosis, 2000. 5:107-114). Later, it wasdiscovered that synthetic duplexes of 21 nucleotide RNAs could mediategene specific RNAi in mammalian cells, without stimulating the genericantiviral defense mechanisms (see Elbashir et al. Nature 2001,411:494-498 and Caplen et al. PNAS USA 2001, 98:9742-9747). As a result,small interfering RNAs (siRNAs) have become powerful tools in attemptingto understand gene function.

RNA interference (RNAi) in mammals is mediated by small interfering RNAs(siRNAs) (Fire et al, Nature 1998, 391:806) or microRNAs (miRNAs)(Ambros, Nature 2004, 431(7006):350-355; Bartel, Cell 2004, 116(2):281-97). The corresponding process in plants is commonly referred to asspecific post-transcriptional gene silencing (PTGS) or RNA silencing andis also referred to as quelling in fungi.

An siRNA is a double-stranded RNA or modified RNA molecule whichdown-regulates or silences (prevents) the expression of a gene/mRNA ofits endogenous (cellular) counterpart.

The selection and synthesis of siRNA corresponding to known genes hasbeen widely reported; (see for example Ui-Tei et al., J Biomed Biotech.2006; 2006: 65052; Chalk et al., BBRC. 2004, 319(1): 264-74; Sioud &Leirdal, Met. Mol Biol.; 2004, 252:457-69; Levenkova et al., Bioinform.2004, 20(3):430-2; Ui-Tei et al., NAR. 2004, 32(3):936-48).

For examples of the use of, and production of, modified siRNA see, forexample, Braasch et al., Biochem. 2003, 42(26):7967-75; Chiu et al.,RNA, 2003, 9(9):1034-48; PCT publications WO 2004/015107 (atugen AG) andWO 02/44321 (Tuschl et al). U.S. Pat. Nos. 5,898,031 and 6,107,094,teach chemically modified oligomers. US Patent Publication Nos.2005/0080246 and 2005/0042647 relate to oligomeric compounds having analternating motif and dsRNA compounds having chemically modifiedinternucleoside linkages, respectively.

Other modifications have been disclosed. The inclusion of a 5′-phosphatemoiety was shown to enhance activity of siRNAs in Drosophila embryos(Boutla, et al., Curr. Biol. 2001, 11:1776-1780) and is required forsiRNA function in human HeLa cells (Schwarz et al., Mol. Cell, 2002,10:537-48). Amarzguioui et al., (NAR, 2003, 31(2):589-95) showed thatsiRNA activity depended on the positioning of the 2′-O-methyl (2′OMe)modifications. Holen et al (NAR. 2003, 31(9):2401-07) report that ansiRNA having small numbers of 2′OMe modified nucleosides gave goodactivity compared to wild type but that the activity decreased as thenumbers of 2′OMe modified nucleosides was increased. Chiu and Rana (RNA.2003, 9:1034-48) teach that incorporation of 2′OMe modified nucleosidesin the sense or antisense strand (fully modified strands) severelyreduced siRNA activity relative to unmodified siRNA. The placement of a2′OMe group at the 5′-terminus on the antisense strand was reported toseverely limit activity whereas placement at the 3′-terminus of theantisense and at both termini of the sense strand was tolerated(Czauderna et al., NAR. 2003, 31(11):2705-16; WO 2004/015107).

Several studies have revealed that siRNA therapeutics are effective invivo in both mammals and in humans. Bitko et al., have shown thatspecific siRNA molecules directed against the respiratory syncytialvirus (RSV) nucleocapsid N gene are effective in treating mice whenadministered intranasally (Nat. Med. 2005, 11(1):50-55). Recent reviewsdiscussing siRNA therapeutics are available (Barik, et al., J. Mol. Med2005, 83:764-773; Dallas and Vlassov, Med. Sci. Monitor 2006,12(4):RA67-74; Chakraborty, Current Drug Targets 2007, 8(3):469-82;Dykxhoorn et al., Gene Therapy 2006. 13:541-552).

Mucke (IDrugs 2007 10(1):37-41) presents a review of currenttherapeutics, including siRNA to various targets, for the treatment ofocular diseases, for example age related macular degeneration (AMD) andglaucoma.

A number of PCT applications have recently been published that relate tothe RNAi phenomenon. These include: PCT publication WO 00/44895; PCTpublication WO 00/49035; PCT publication WO 00/63364; PCT publication WO01/36641; PCT publication WO 01/36646; PCT publication WO 99/32619; PCTpublication WO 00/44914; PCT publication WO 01/29058; and PCTpublication WO 01/75164.

RNA interference (RNAi) is based on the ability of dsRNA species toenter a cytoplasmic protein complex, where it is then targeted to thecomplementary cellular RNA and specifically degrade it. The RNAinterference response features an endonuclease complex containing ansiRNA, commonly referred to as an RNA-induced silencing complex (RISC),which mediates cleavage of single-stranded RNA having a sequencecomplementary to the antisense strand of the siRNA duplex. Cleavage ofthe target RNA may take place in the middle of the region complementaryto the antisense strand of the siRNA duplex (Elbashir et al., GenesDev., 2001, 15(2):188-200). In more detail, longer dsRNAs are digestedinto short (17-29 bp) dsRNA fragments (also referred to as shortinhibitory RNAs, “siRNAs”) by type III RNAses (DICER, DROSHA, etc.;Bernstein et al., Nature, 2001, 409(6818):363-6; Lee et al., Nature,2003, 425(6956):415-9). The RISC protein complex recognizes thesefragments and complementary mRNA. The whole process is culminated byendonuclease cleavage of target mRNA (McManus & Sharp, Nature Rev Genet,2002, 3(10):737-47; Paddison & Hannon, Curr Opin Mol Ther. 2003,5(3):217-24). (For additional information on these terms and proposedmechanisms, see for example Bernstein et al., RNA 2001, 7(11):1509-21;Nishikura, Cell 2001, 107(4):415-8 and PCT publication WO 01/36646).

siRNA Structures

The selection and synthesis of siRNA corresponding to known genes hasbeen widely reported; (see for example Ui-Tei et al., J Biomed Biotech.2006; 2006: 65052; Chalk et al., BBRC. 2004, 319(1): 264-74; Sioud &Leirdal, Met. Mol Biol.; 2004, 252:457-69; Levenkova et al., Bioinform.2004, 20(3):430-2; Ui-Tei et al., NAR. 2004, 32(3):936-48).

For examples of the use of, and production of, modified siRNA see, forexample, Braasch et al., Biochem. 2003, 42(26):7967-75; Chiu et al.,RNA, 2003, 9(9):1034-48; PCT publications WO 2004/015107 (atugen AG) andWO 02/44321 (Tuschl et al). U.S. Pat. Nos. 5,898,031 and 6,107,094,teach chemically modified oligomers. US Patent Publication Nos.2005/0080246 and 2005/0042647 relate to oligomeric compounds having analternating motif and dsRNA compounds having chemically modifiedinternucleoside linkages, respectively.

Other modifications have been disclosed. The inclusion of a 5′-phosphatemoiety was shown to enhance activity of siRNAs in Drosophila embryos(Boutla, et al., Curr. Biol. 2001, 11:1776-1780) and is required forsiRNA function in human HeLa cells (Schwarz et al., Mol. Cell, 2002,10:537-48). Amarzguioui et al., (NAR, 2003, 31(2):589-95) showed thatsiRNA activity depended on the positioning of the 2′-O-methyl (2′OMe)modifications. Holen et al (NAR. 2003, 31(9):2401-07) report that ansiRNA having small numbers of 2′OMe modified nucleosides gave goodactivity compared to wild type but that the activity decreased as thenumbers of 2′OMe modified nucleosides was increased. Chiu and Rana (RNA.2003, 9:1034-48) teach that incorporation of 2′OMe modified nucleosidesin the sense or antisense strand (fully modified strands) severelyreduced siRNA activity relative to unmodified siRNA. The placement of a2′OMe group at the 5′-terminus on the antisense strand was reported toseverely limit activity whereas placement at the 3′-terminus of theantisense and at both termini of the sense strand was tolerated(Czauderna et al., NAR. 2003, 31(11):2705-16; WO 2004/015107).

The double stranded RNA molecules disclosed herein possess one 3′non-nucleotide overhang on either the sense or antisense strand andoptionally two 3′ overhangs, one on each of the sense and antisensestrands.

The molecules disclosed herein offer an advantage in that they arenon-toxic and are useful in the preparation of pharmaceuticalcompositions for treatment of various diseases and disorders.

PCT Patent Application No. PCT/IL2007/001278 (PCT Publication No. WO2008/050329) and U.S. Ser. No. 11/978,089 to the assignee of the presentinvention relate to inhibitors of pro-apoptotic genes, and areincorporated by reference in their entirety.

The present invention relates generally to compounds which down-regulateexpression of various genes, particularly to novel small interferingRNAs (siRNAs), and to the use of these novel siRNAs in the treatment ofa subject suffering from various medical conditions.

Molecules and compositions are discussed herein at length, and any ofsaid molecules and/or compositions may be beneficially employed in thetreatment of a subject suffering from any of said conditions.

The siRNA compounds of the present invention possess structures andmodifications which may for example increase activity, increasestability, and or minimize toxicity; the novel modifications of thesiRNAs of the present invention are beneficially applied to doublestranded RNA useful in preventing or attenuating target gene expression,in particular the target genes discussed herein.

According to one aspect provided herein are inhibitory oligonucleotidecompounds comprising unmodified and/or modified nucleotides. One strandof the compound comprises at least one 3′ overhang comprising at leastone non-nucleotide moiety, preferably two non-nucleotide moieties. Thecompounds disclosed herein preferably comprise unmodifiedribonucleotides and modified ribonucleotides and or one or moreunconventional moiety. In some embodiments at least one of N or N′ isselected from the group consisting of a sugar modification, a basemodification and an internucleotide linkage modification. In someembodiments the compounds disclosed herein include at least one modifiednucleotide including DNA, LNA (locked nucleic acid) including ENA(ethylene-bridged nucleic acid; PNA (peptide nucleic acid); arabinoside;PACE (phosphonoacetate and derivatives thereof), mirror nucleotide, ornucleotides with a 6 member sugar analog (e.g. hexose or morpholino).

In one embodiment the compound comprises at least one modifiedribonucleotide having a 2′ modification on the sugar moiety (“2′ sugarmodification”). In certain embodiments the compound comprises 2′O-alkylor 2′-fluoro or 2′O-allyl or any other 2′ sugar modification, optionallyon alternate positions. One possible 2′ modification is 2′ O-methyl (2′methoxy, 2′ OMe).

Other stabilizing modifications are also possible (e.g. modifiednucleotides added to a 3′ or 5′ terminus of an oligomer). In someembodiments the backbone of the oligonucleotides is modified andcomprises phosphate-D-ribose entities but may also containthiophosphate-D-ribose entities, phosphodiester L-ribose entities,triester, thioate, 2′-5′ bridged backbone (also may be referred to as5′-2′), PACE modified internucleotide linkage or any other type ofmodification.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology used is intended to be in the natureof words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventioncan be practiced otherwise than as specifically described.

The present invention is illustrated in detail below with reference toexamples, but is not to be construed as being limited thereto.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theclaimed invention in any way.

Standard molecular biology protocols known in the art not specificallydescribed herein are generally followed essentially as in Sambrook etal., Molecular cloning: A laboratory manual, Cold Springs HarborLaboratory, New-York (1989, 1992), and in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1988), and as in Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1989) and as in Perbal, APractical Guide to Molecular Cloning, John Wiley & Sons, New York(1988), and as in Watson et al., Recombinant DNA, Scientific AmericanBooks, New York and in Birren et al (eds) Genome Analysis: A LaboratoryManual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York(1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828;4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein byreference. Polymerase chain reaction (PCR) was carried out generally asin PCR Protocols: A Guide To Methods And Applications, Academic Press,San Diego, Calif. (1990). In situ (In cell) PCR in combination with FlowCytometry is useful for detection of cells containing specific DNA andmRNA sequences (Testoni et al., Blood 1996, 87:3822.) Methods ofperforming RT-PCR are also well known in the art.

SEQUENCE LISTING

The Sequence Listing filed electronically herewith is herebyincorporated by reference in its entirety (File Name: 217_PCT2_ST25.txt;Date Created: Jan. 6 2011; File Size: 6.00 Kb.)

Example 1 Generation of Nucleic Acid Molecules and In Vitro Testing ofModified siRNA Compounds

Using proprietary algorithms and the known sequence of a targetnucleotide, for example the mRNA of a target gene, the sense andantisense sequences of many potential siRNA nucleic acid molecules aregenerated. Nucleic acid molecules are depicted in 5′ to 3′ orientation,and the sense and complementary antisense sequences are depicted on thesame line in the tables, unless otherwise noted.

Table A provides exemplary, non-limiting, nucleic acid sequences usefulin generating nucleic acid molecules disclosed herein.

TABLE A SEQ SEQ ID ID Sense 5′>3′ NO Antisense 5′>3′ NO CASP2_GCCAGAAUGUGGAACUCCU 1 AGGAGUUCCACAUUCUGGC 2  4 MYD88_GAAUGUGACUUCCAGACCA 3 UGGUCUGGAAGUCACAUUC 4 11 RAC1_ GAGUCCUGCAUCAUUUGAA5 UUCAAAUGAUGCAGGACUC 6  2 RHOA_ UCGACAGCCCUGAUAGUUU 7AAACUAUCAGGGCUGUCGA 8 29 RHOA_ CAGAAGUCAUCUUGCUACA 9 UGUAGCAAGAUGACUUCUG10 48 RHOA_ GUGGCAGAGUUACAGUUCA 11 UGAACUGUAACUCUGCCAC 12 50 RHOA_GUGGCAGAGUUACAGUUCU 13 AGAACUGUAACUCUGCCAC 14 58 RHOA_CAUCGACAGCCCUGAUAGA 15 UCUAUCAGGGCUGUCGAUG 16 60 RHOA_GAUCUUCGGAAUGAUGAGA 17 UCUCAUCAUUCCGAAGAUC 18 61 RHOA_CAUCGACAGCCCUGAUAGU 19 ACUAUCAGGGCUGUCGAUG 20 70 RHOA_UCGACAGCCCUGAUAGUUA 21 UAACUAUCAGGGCUGUCGA 22 75 TLR2_GGUGGAGAACCUUAUGGUC 23  GACCAUAAGGUUCUCCACC 24 37 TLR2_AGAUAAUGAACACCAAGAC 25  GUCUUGGUGUUCAUUAUCU 26 46 CASP2_GAAUGUGGAACUCCUCAAC 27  GUUGAGGAGUUCCACAUUC 28 25

Activity:

Single stranded oligonucleotides (sense strand and antisense strand) aresynthesized using standard synthesis procedures. DMT-propane-Diolphosphoramidite ChemGenes; CLP-9908) is coupled at a concentration of0.05M. Duplexes are generated by annealing complementary single strandedoligonucleotides. In a laminar flow hood, a ˜500 μM Stock Solution ofsingle stranded oligonucleotide is prepared by diluting in WFI (waterfor injection, Norbrook). Actual ssRNA concentrations are determined bydiluting each 500 μM ssRNA 1:200 using WFI, and measuring the OD usingNano Drop. The procedure is repeated 3 times and the averageconcentration is calculated. The Stock Solution was then diluted to afinal concentration of 250 μM. Complementary single strands wereannealed by heating to 85° C. and allowing to cool to room temperatureover at least 45 minutes. Duplexes were tested for complete annealing bytesting 5 μl on a 20% polyacrylamide gel and staining. Samples werestored at −80° C.

The double stranded nucleic acid molecules disclosed herein were testedfor activity as follows: About 1.5-2×10⁵ tested cells (HeLa cells and/or293T cells for siRNA targeting human genes and NRK52 (normal rat kidneyproximal tubule cells) cells and/or NMuMG cells (mouse mammaryepithelial cell line) for siRNA targeting the rat/mouse gene) wereseeded per well in 6 wells plate (70-80% confluent).

About 24 hours later, cells were transfected with modified siRNAcompounds using the Lipofectamine™ 2000 reagent (Invitrogen) at finalconcentrations of from 0.001 μM to about 50 μM. The cells were incubatedat 37° C. in a CO₂ incubator for 72 h.

As positive control for transfection PTEN-Cy3 labeled modified siRNAcompounds are used. GFP siRNA compounds are used as negative control forsiRNA activity.

At 72 h after transfection cells are harvested and RNA was extractedfrom cells. Transfection efficiency is tested by fluorescent microscopy.

The percent of inhibition of gene expression using specific preferredsiRNA structures is determined using qPCR analysis of a target gene incells expressing the endogenous gene.

The IC50 value of the tested RNAi activity is determined by constructinga dose-response curve using the activity results obtained with thevarious final siRNA concentrations. The dose response curve isconstructed by plotting the relative amount of residual target mRNAversus the logarithm of transfected siRNA concentration. The curve iscalculated by fitting the best sigmoid curve to the measured data. Themethod for the sigmoid fit is also known as a 3-point curve fit.

$Y = {{Bot} + \frac{100 - {Bot}}{1 + 10^{{({{{LogIC}\; 50} - X})} \times {HillSlope}}}}$

where Y is the residual target mRNA response, X is the logarithm oftransfected siRNA concentration, Bot is the Y value at the bottomplateau, Log IC50 is the X value when Y is halfway between bottom andtop plateaus and HillSlope is the steepness of the curve.

Serum Stability Experiments

The double stranded nucleic acid molecules were tested for duplexstability in human serum or human tissue extract, as follows:

siRNA molecules at final concentration of 7 uM are incubated at 37° C.in 100% human serum (Sigma Cat# H4522). (siRNA stock 100 uM diluted inhuman serum 1:14.29 or human tissue extract from various tissue types.).Five ul (5 ul) are added to 15 ul 1.5×TBE-loading buffer at differenttime points (for example 0, 30 min, 1 h, 3 h, 6 h, 8 h, 10 h, 16 h and24 h). Samples are immediately frozen in liquid nitrogen and are kept at−20° C.

Each sample is loaded onto a non-denaturing 20% acrylamide gel, preparedaccording to methods known in the art. The oligos are visualized withethidium bromide under UV light.

In general, the siRNAs having specific sequences that are selected forin vitro testing are specific for human and a second species such as rator rabbit genes.

Stability to Exonucleases

To study the stabilization effect of 3′ non-nucleotide moieties on anucleic acid molecule the sense strand, the antisense strand and theannealed siRNA duplex are incubated in cytosolic extracts prepared fromdifferent cell types. A protocol for testing stability in HCT116 cellsis provided below.

Extract: HCT116 cytosolic extract (12 mg/ml).

Extract buffer: 25 mM Hepes pH-7.3 at 37° C.; 8 mM MgCl; 150 mM NaClwith 1 mM DTT was added fresh immediately before use.

Method: 3.5 ml of test siRNA (100 mM), were mixed with 46.5 ml contain120 mg of HCT116 cytosolic extract. The 46.5 ml consists of 12 ml ofHCT116 extract, and 34.5 ml of the extract buffer supplemented with DTTand protease inhibitors cocktail/100 (Calbiochem, setIII-539134). Thefinal concentration of the siRNA in the incubation tube is 7 mM. Thesample was incubated at 37° C., and at the indicated time point 5 mlwere moved to fresh tube, mixed with 15 ml of 1×TBE-50% Glycerol loadingbuffer, and snap frozen in Liquid N2. The final concentration of thesiRNA in the loading buffer is 1.75 mM (21 ng siRNA/ml). For Analyses bynative PAGE and EtBr staining 50 ng are loaded per lane. For Northernanalyses ing of tested siRNA was loaded per lane. Other cell typesinclude HeLa and hepatic stellate cells (HSC).

The applicants have shown that nucleic acid molecules which include the3′ terminal alkyl; or alkyl derivative overhang exhibit enhancedstability compared to a blunt ended nucleic acid molecules and nucleicacid molecules comprising 3′ nucleotide overhangs.

Exemplary Compounds

siRNA compounds comprising non-nucleotide moieties covalently attachedto the 3′ terminus were synthesized and tested as described above. FIG.2 provides a table of compounds useful in RNAi comprising sequences andmodifications disclosed herein. Legend for the modifications follows: aprefix “z” indicates a moiety (nucleotide or non-nucleotide) covalentlyattached to the 3′ or 5′ terminal nucleotide. For example zdT refers toa dT overhang; zdT; zdT refers to a dTdT overhang. A prefix “y”indicates a nucleotide substitution, for example yLdA refers to aL-deoxyriboadenine substituted for a ribonucleotide in the sense strandor antisense strand; and ydT refers to a deoxyribothymidine substitutedfor a ribonucleotide in the sense or antisense oligonucleotide. A prefix“m” refers to a 2′OMe sugar modified ribonucleotide. Additional codesare set forth hereinbelow in Table B.

TABLE B Code Description Ra riboadenosine-3′-phosphate; 3′-adenylic acidRC ribocytidine-3′-phosphate; 3′-cytidylic acid RGriboguanosine-3′-phosphate; 3′-guanylic acid RUribouridine-3′-phosphate; 3′-uridylic acid mA2′-O-methyladenosine-3′-phosphate; 2′-O-methyl-3′-adenylic acid mC2′-O-methylcytidine-3′-phosphate; 2′-O-methyl-3′-cytidylic acid mG2′-O-methylguanosine-3′-phosphate; 2′-O-methyl-3′-guanylic acid mU2′-O-methyluridine-3′-phosphate; 2′-O-methyl-3′-uridylic acid dAdeoxyriboadenosine-3′-phosphate; 2′-deoxyribo-3′-adenylic acid dCdeoxyribocytidine-3′-phosphate; 2′-deoxyribo-3′-cytidylic acid dGdeoxyriboguanosine-3′-phosphate; 2′-deoxyribo-3′-guanylic acid dTthymidine-3′-phosphate; 3′-thymidylic acid rA2priboadenosine-2′-phosphate; 2′-adenylic acid rC2pribocytidine-2′-phosphate; 2′-cytidylic acid rG2priboguanosine-2′-phosphate; 2′-guanylic acid rU2pribouridine-2′-phosphate; 2′-uridylic acid LdAL-deoxyriboadenosine-3′-phosphate (mirror image dA) LdCL-deoxyribocytidine-3′-phosphate (mirror image dC) LdGL-deoxyriboguanosine-3′-phosphate (mirror image dG) LdTL-deoxyribothymidine-3′-phosphate (mirror image dT) DB abasicdeoxyribose-3′-phosphate; 1,2-dideoxy-D-ribofuranose-3- phosphate;1,4-anhydro-2-deoxy-D-ribitol-3-phosphate zidB Inverted abasicdeoxyribose-5′-phosphate at terminus; 5′ = 5′-5′ idAb; At 3′ = 3′-3′idAb P 5′ phosphate S 5′ phosphorothioate $ lacking a 3′ linker (usedtogether with above nucleotides at the 3′ end of the sequence) 3mN2p3′-O-methyl ribo-nucleotide-2′-phosphate yC3p substitute aribonucleotide with 3-Hydroxypropane-1-phosphate ydA substitute aribonucleotide with deoxyriboAdenosine-3′-phosphate; ydT substitute aribonucleotide with deoxyriboThymidine-3′-phosphate; ydU substitute aribonucleotide with deoxyUridine yLdA substitute a ribonucleotide withL-deoxyriboAdenosine-3′-phosphate yLdC substitute a ribonucleotide withL-deoxyriboCytidine-3′-phosphate yLdG substitute a ribonucleotide withL-deoxyriboGuanosine-3′-phosphate ymA substitute a ribonucleotide with2′-O-methylAdenosine-3′-phosphate; ymC substitute a ribonucleotide with2′-O-methylCytidine-3′-phosphate; ymU substitute a ribonucleotide with2′-O-methylUridine-3′-phosphate; yrA substitute a ribonucleotide withriboAdenosine-3′-phosphate; yrC substitute a ribonucleotide withriboCytidine-3′-phosphate; yrG substitute a ribonucleotide withriboGuanosine-3′-phosphate; yrU substitute a ribonucleotide withriboUridine-3′-phosphate; zC3p (CH2)3-Pi = 3-Hydroxypropane-1-phosphate(C3Pi) zC3p; zC3p (CH2)3-Pi x2; = 3-Hydroxypropane-1-phosphate;(C3Pi-C3Pi) zc3p; zc3p; zc3p (CH2)3-Pi x3; =3-Hydroxypropane-1-phosphate; (C3Pi-C3Pi-C3Pi) zc3p; zc3ps (CH2)3-Pi;(CH2)-3′phosphorothioate (C3Pi-C3Ps) zC3p; zrB (CH2)3-Pi;ribo-Abasic-3′-Pi zC3p; zrG (CH2)3-Pi_rG zC6Np Amino-C6-Phosphate zC6Np;zrC; zrA Amino-C6-Phosphate_rCrA zdB; zdB abasicdeoxyribose-3′-phosphate x2 (dAb-dAb) ZdT Deoxy-Thymidine-3′-PhosphatezdT; zdT dTdT overhang at 3′ ZidB Inverted abasicdeoxyribose-5′-phosphate; At 5′ = 5′-5′ idAb; At 3′ = 3′- 3′ idAb ZidTInverted-Deoxy-Thymidine-5′-Phosphate ZiLd Inverted L-DNA ZirB Invertedabasic ribose-5′-phosphate zirB; zirB Inverted abasicribose-5′-phosphate x2 zirB; zrC; zrA Inverted abasicribose-3′-phosphate_rCrA ZLdA L-deoxyriboAdenosine-3′-phosphate ZLdCL-deoxyriboCytidine-3′-phosphate ZLdT L-deoxyriboThymidine-3′-phosphateZmC 2′-O-methylcytidine-3′-ethoxyphosphate ZmU2′-O-methyluridine-3′-ethoxyphosphate ZOle Oleic acid zrA; zrG rArG zrB;zrB abasic ribose-3′-phosphate x2 zrC; zrA rC; rA zrU; zrG rUrG zrU; zrUrUrU

In the following tables, the codes used in the column labeled “Sense5->3 Antisense 5->3” are the codes shown in Table B. The columns labeledas “sense modifications” and “antisense modifications” provide a briefdescription of the positional modifications used in each of the senseand antisense strands, for example 20-C3; C3 refers to a 3′ C3Pi-C3OHterminal overhang and 20-dTdT refers to a 3′ dTdT terminal overhang(beginning at position 20 on a 19-mer), 2,4,6,8,10,12,14,16,18-2′-OMerefers to 2′OMe sugar modified ribonucleotides in positions 2, 4, 6, 8,10, 12, 14, 16, 18.

TABLE 1 stability compound in hHSC Sense 5−>3 sense antisense No. nameextract Antisense 5−>3 modifications modifications 1 RAC1_2_S710 <3 hrG; rA; rG; rU; rC; rC; rU; rG; rC; — — rA; rU; rC; rA; rU; rU; rU; rG;rA; rA$ rU; rU; rC; rA; rA; rA; rU; rG; rA; rU; rG; rC; rA; rG; rG; rA;rC; rU; rC$ 2 RAC1_2_S1231 6 h-12 h rG; rA; rG; rU; rC; rC; rU; rG; rC;20-C3; C3 20-C3; C3 rA; rU; rC; rA; rU; rU; rU; rG; rA; rA; zc3p; zc3p$rU; rU; rC; rA; rA; rA; rU; rG; rA; rU; rG; rC; rA; rG; rG; rA; rC; rU;rC; zc3p; zc3p$ 3 RAC1_2_S709 6 h-12 h rG; rA; rG; rU; rC; rC; rU; rG;rC; 20-dTdT 20-dTdT rA; rU; rC; rA; rU; rU; rU; rG; rA; rA; zdT; zdT$rU; rU; rC; rA; rA; rA; rU; rG; rA; rU; rG; rC; rA; rG; rG; rA; rC; rU;rC; zdT; zdT$ 4 RAC1_2_S1759 6 h-12 h rG; rA; rG; rU; rC; rC; rU; rG;rC; 20-mU; mU 20-mU; mU rA; rU; rC; rA; rU; rU; rU; rG; rA; rA; zmU;zmU$ rU; rU; rC; rA; rA; rA; rU; rG; rA; rU; rG; rC; rA; rG; rG; rA; rC;rU; rC; zmU; zmU$

The data presented in Table 1 shows that C3Pi-C3OH 3′ terminal overhang(2) improves siRNA nuclease resistance in cell extracts compared toblunt unmodified siRNA (1). The stabilizing effect of the C3-C3 issimilar to the dTdT (3) and 2′OMe U (4) 3′ terminal overhangs. RAC1_(—)2sequences are set forth in SEQ ID NOS:5 and 6.

TABLE 2 stability of compound sense strand Patent Sense 5−>3 senseantisense No name in HCT116 Antisense 5−>3 modifications modifications 1CASP2_4_S1152 24 h rG; rC; rC; rA; rG; rA; rA; rU; rG; rU; 20-C3; C3 —rG; rG; rA; rA; rC; rU; rC; rC; rU; zc3p; (unmodified) zc3p$ rA; rG; rG;rA; rG; rU; rU; rC; rC; rA; rC; rA; rU; rU; rC; rU; rG; rG; rC$ 2CASP2_4_S1153 24 h zidB; rG; rC; rC; rA; rG; rA; rA; rU; rG;0,20-cap-idAb rU; rG; rG; rA; rA; rC; rU; rC; rC; rU; zidB$ rA; rG; rG;rA; rG; rU; rU; rC; rC; rA; rC; rA; rU; rU; rC; rU; rG; rG; rC$ 3CASP2_4_S710  1 h rG; rC; rC; rA; rG; rA; rA; rU; rG; rU; — — rG; rG;rA; rA; rC; rU; rC; rC; rU$ rA; rG; rG; rA; rG; rU; rU; rC; rC; rA; rC;rA; rU; rU; rC; rU; rG; rG; rC$

The data in Table 2 shows that C3-C3 3′ terminal overhang (1) and astrand comprising 5′ and 3′ inverted deoxy abasic moieties (2) improvesstrand nuclease resistance in cell extract when compared to unmodifiedstrand (3). CASP2_(—)4 sequences are set forth in SEQ ID NOS:1 and 2.

TABLE 3 stability in Compound IC50, cell extract, Sense 5−>3 senseantisense No. name nM hours (FIG. 4) Antisense 5−>3 modificationsmodifications 1 RAC1_2_S73 0.207 rG; mA; rG; mU; rC; mC; rU; mG; rC;2,4,6,8,10,12, 1,3,5,7,9,11,13, mA; rU; mC; rA; mU; rU; mU; rG; mA;14,16,18-2′- 15,17,19-2′- rA$ OMe-3′-Pi OMe-3′-Pi mU; rU; mC; rA; mA;rA; mU; rG; mA; rU; mG; rC; mA; rG; mG; rA; mC; rU; mC$ 2 RAC1_2_S10050.067 rG; mA; rG; mU; rC; mC; rU; mG; rC; 2,4,6,8,10,12,1,3,5,7,9,11,13, mA; rU; mC; rA; mU; rU; mU; rG; mA; 14,16,18-2′-15,17,19-2′- rA; zidB$ OMe-3′-Pi; 20- OMe-3′-Pi; 20- mU; rU; mC; rA; mA;rA; mU; rG; mA; cap-idAb cap-idAb rU; mG; rC; mA; rG; mG; rA; mC; rU;mC; zidB$ 3 RAC1_2_S1154 0.082 rG; mA; rG; mU; rC; mC; rU; mG; rC;2,4,6,8,10,12, 1,3,5,7,9,11,13, mA; rU; mC; rA; mU; rU; mU; rG; mA;14,16,18-2′- 15,17,19-2′- rA; zc3p; zc3p$ OMe-3′-Pi; 20- OMe-3′-Pi; 20-mU; rU; mC; rA; mA; rA; mU; rG; mA; C3; C3 C3; C3 rU; mG; rC; mA; rG;mG; rA; mC; rU; mC; zc3p; zc3p$ 4 RAC1_2_S1156 0.174 rG; mA; rG; mU; rC;mC; rU; mG; rC; 2,4,6,8,10,12, 1,3,5,7,9,11,13, mA; rU; mC; rA; mU; rU;mU; rG; mA; 14,16,18-2′- 15,17,19-2′- rA$ OMe-3′-Pi OMe-3′-Pi; 20- mU;rU; mC; rA; mA; rA; mU; rG; mA; C3; C3 rU; mG; rC; mA; rG; mG; rA; mC;rU; mC; zc3p; zc3p$ 5 CASP2_4_S505 1.1 36 rG; rC; rC; rA; rG; rA; rA;rU; rG; rU; 18-L-DNA-3′-Pi 2,4,6,8,11,13, rG; rG; rA; rA; rC; rU; rC;LdC; rU$ 15,17,19-2′- rA; mG; rG; mA; rG; mU; rU; mC; rC; OMe-3′-Pi rA;mC; rA; mU; rU; mC; rU; mG; rG; mC$ 6 CASP2_4_S796 0.202 rG; rC; rC; rA;rG; rA; rA; rU; rG; rU; 18-L-DNA-3′-Pi 2,4,6,8,11,13, rG; rG; rA; rA;rC; rU; rC; LdC; rU$ 15,17,19-2′- rA; mG; rG; mA; rG; mU; rU; mC; rC;OMe-3′-Pi; 20- rA; mC; rA; mU; rU; mC; rU; mG; rG; dTdT mC; zdT; zdT$ 7CASP2_4_S800 0.169 36 rG; rC; rC; rA; rG; rA; rA; rU; rG; rU;18-L-DNA-3′-Pi 2,4,6,8,11,13, rG; rG; rA; rA; rC; rU; rC; LdC; rU$15,17,19-2′- rA; mG; rG; mA; rG; mU; rU; mC; rC; OMe-3′-Pi; 20- rA; mC;rA; mU; rU; mC; rU; mG; rG; C3; C3 mC; zc3p; zc3p$ 8 CASP2_4_S802 0.216rG; rC; rC; rA; rG; rA; rA; rU; rG; rU; 18-L-DNA-3′-Pi 2,4,6,8,11,13,rG; rG; rA; rA; rC; rU; rC; LdC; rU$ 15,17,19-2′- rA; mG; rG; mA; rG;mU; rU; mC; rC; OMe-3′-Pi; 20- rA; mC; rA; mU; rU; mC; rU; mG; rG; C3;rAb mC; zc3p; zrB$

The data provided in Table 3 shows that 1) introduction of C3-C3moieties (3, 4 and 7), inverted deoxy abasic (2), C3-ribo abasic (8) at3′ end of antisense or on both antisense and sense strand improves siRNAactivity compared to a blunt ended compounds (1 and 5) and is moreactive than the same compound with 3′ dTdT overhangs (6); and that 2)the improved activity of the compound comprising the 3′ terminal C3-C3is not due to increased stability in cell extract since both compounds(5 and 7) are highly stable (at least 36 h).

TABLE 4 % Residual Compound concen- target Sense 5->3 sense antisenseNo. name tration mRNA Antisense 5->3 modifications modifications 1RAC1_2_ 40 nM 25 rG; mA; rG; mU; rC; mC; rU; mG; rC; 2, 4, 6, 8, 10, 1,3, 5, 7, 9, 11, 13, S73  5 nM 35 mA; rU; mC; rA; mU; rU; mU; rG; 12, 14,16, 18- 15, 17, 19-2′-OMe- mA; rA$ 2′-OMe-3′- 3′-Pi mU; rU; mC; rA; mA;rA; mU; rG; Pi mA; rU; mG; rC; mA; rG; mG; rA; mC; rU; mC$ 2 RAC1_2_ 40nM 12 rG; mA; rG; mU; rC; mC; rU; mG; rC; 2, 4, 6, 8, 10, 1, 3, 5, 7, 9,11, 13, S1154  5 nM 32 mA; rU; mC; rA; mU; rU; mU; rG; 12, 14, 16, 18-15, 17, 19-2′-OMe- mA; rA; zc3p; zc3p$ 2′-OMe-3′- 3′-Pi; 20-C3; C3 mU;rU; mC; rA; mA; rA; mU; rG; Pi; 20-C3; C3 mA; rU; mG; rC; mA; rG; mG;rA; mC; rU; mC; zc3p; zc3p$ 3 RAC1_2_ 40 nM 32 rG; mA; rG; mU; rC; mC;rU; mG; rC; 2, 4, 6, 8, 10, 1, 3, 5, 7, 9, 11, 13, S1155  5 nM 50 mA;rU; mC; rA; mU; rU; mU; rG; 12, 14, 16, 18- 15, 17, 19-2′-OMe- mA; rA;zc3p; zc3p$ 2′-OMe-3′- 3′-Pi mU; rU; mC; rA; mA; rA; mU; rG; Pi; 20-C3;C3 mA; rU; mG; rC; mA; rG; mG; rA; mC; rU; mC$ 4 RAC1_2_ 40 nM 24 rG;mA; rG; mU; rC; mC; rU; mG; rC; 2, 4, 6, 8, 10, 1, 3, 5, 7, 9, 11, 13,S1156  5 nM 44 mA; rU; mC; rA; mU; rU; mU; rG; 12, 14, 16, 18- 15, 17,19-2′-OMe- mA; rA$ 2′-OMe-3′- 3′-Pi; 20-C3; C3 mU; rU; mC; rA; mA; rA;mU; rG; Pi mA; rU; mG; rC; mA; rG; mG; rA; mC; rU; mC; zc3p; zc3p$

Data in Table 4 shows that the most pronounced activity increase isobtained when C3C3 is present at the 3′ termini of both antisense andsense strands (2) compared to blunt (1) and to C3-C3 on only one of thestrands (3 or 4).

TABLE 5 % Residual Compound Concen- target Sense 5->3 sense antisenseNo. name tration mRNA Antisense 5->3 modifications modifications 1CASP2_4_ 20 nM 30 rG; rC; rC; rA; rG; rA; rA; rU; 18-L-DNA-3′-Pi 2, 4,6, 8, 11, 13, S953  5 nM 17 rG; rU; rG; rG; rA; rA; rC; rU; 15, 17,19-2′-  1 nM 13 rC; LdC; rU$ OMe-3′-Pi; 20- rA; mG; rG; mA; rG; mU; rU;C3; C3; Phosphate mC; rC; rA; mC; rA; mU; rU; mC; rU; mG; rG; mC; zc3p;zc3p 2 CASP2_4_ 20 nM 65 rG; rC; rC; rA; rG; rA; rA; rU; 18-L-DNA-3′-Pi2, 4, 6, 8, 11, 13, S1145  5 nM 43 rG; rU; rG; rG; rA; rA; rC; rU; 15,17, 19-2′-  1 nM 26 rC; LdC; rU$ OMe-3′-Pi; 20- rA; mG; rG; mA; rG; mU;rU; C3 mC; rC; rA; mC; rA; mU; rU; mC; rU; mG; rG; mC; zc3p$ 3 CASP2_4_20 nM 57 rG; rC; rC; rA; rG; rA; rA; rU; 18-L-DNA-3′-Pi 2, 4, 6, 8, 11,13, S1146  5 nM 43 rG; rU; rG; rG; rA; rA; rC; rU; 15, 17, 19-2′-  1 nM26 rC; LdC; rU$ OMe-3′-Pi; 20- rA; mG; rG; mA; rG; mU; rU; C3; C3; C3mC; rC; rA; mC; rA; mU; rU; mC; rU; mG; rG; mC; zc3p; zc3p; zc3p$ 4CASP2_4_ 20 nM 48 rG; rC; rC; rA; rG; rA; rA; rU; 18-L-DNA-3′-Pi 2, 4,6, 8, 11, 13, S1147  5 nM 26 rG; rU; rG; rG; rA; rA; rC; rU; 15, 17,19-2′-  1 nM 13 rC; LdC; rU$ OMe-3′-Pi; 20- rA; mG; rG; mA; rG; mU; rU;C3; C3-3′ps; mC; rC; rA; mC; rA; mU; rU; Phosphate mC; rU; mG; rG; mC;zc3p; zc3ps 5 CASP2_4_ 20 nM 52 rG; rC; rC; rA; rG; rA; rA; rU;18-L-DNA-3′-Pi 2, 4, 6, 8, 11, 13, S505  5 nM 48 rG; rU; rG; rG; rA; rA;rC; rU; 15, 17, 19-2′-OMe-  1 nM 22 rC; LdC; rU$ 3′-Pi rA; mG; rG; mA;rG; mU; rU; mC; rC; rA; mC; rA; mU; rU; mC; rU; mG; rG; mC$

Data in Table 5 shows that 1) the presence of a C3OH (2) orC3Pi-C3Pi-C3OH (3) moiety on the antisense strand does not improveactivity compared to blunt siRNA (5) and 2) the presence of C3Pi-C3Pi(1) and C3Pi-C3Ps (4) moieties on the antisense strand improves activitycompared to blunt ended compounds and the effect is more pronounced withC3Pi-C3Pi.

TABLE 6 Residual target Compound concen- mRNA Sense 5->3 sense antisenseNo. name tration % Antisense 5->3 modifications modifications 1CASP2_25_ 50 15 rG; mA; rA; mU; rG; mU; rG; mG; rA; 2, 4, 6, 8, 10, 1,3, 5, 7, 9, 11, 13, S1005 20 35 mA; rC; mU; rC; mC; rU; mC; rA; mA; 12,14, 16, 18- 15, 17, 19-2′-  5 45 rC; zidB$ 2′-OMe-3′- OMe-3′-Pi; 20- mG;rU; mU; rG; mA; rG; mG; rA; mG; Pi; 20-cap- cap-idAb rU; mU; rC; mC; rA;mC; rA; mU; rU; idAb mC; zidB$ 2 CASP2_25_ 50 38 rG; mA; rA; mU; rG; mU;rG; mG; rA; 2, 4, 6, 8, 10, 1, 3, 5, 7, 9, 11, 13, S1006 20 38 mA; rC;mU; rC; mC; rU; mC; rA; mA; 12, 14, 16, 18- 15, 17, 19-2′-  5 81 rC;zidB$ 2′-OMe-3′- OMe-3′-Pi mG; rU; mU; rG; mA; rG; mG; rA; mG; Pi;20-cap- rU; mU; rC; mC; rA; mC; rA; mU; rU; idAb mC$ 3 CASP2_25_ 50 24rG; mA; rA; mU; rG; mU; rG; mG; rA; 2, 4, 6, 8, 10, 1, 3, 5, 7, 9, 11,13, S1007 20 46 mA; rC; mU; rC; mC; rU; mC; rA; mA; 12, 14, 16, 18- 15,17, 19-2′-  5 96 rC$ 2′-OMe-3′- OMe-3′-Pi; 20- mG; rU; mU; rG; mA; rG;mG; rA; mG; Pi cap-idAb rU; mU; rC; mC; rA; mC; rA; mU; rU; mC; zidB$ 4CASP2_25_ 50 20 rG; mA; rA; mU; rG; mU; rG; mG; rA; 2, 4, 6, 8, 10, 1,3, 5, 7, 9, 11, 13, S73 20 56 mA; rC; mU; rC; mC; rU; mC; rA; mA; 12,14, 16, 18- 15, 17, 19-2′-  5 64 rC$ 2′-OMe-3′- OMe-3′-Pi mG; rU; mU;rG; mA; rG; mG; rA; mG; Pi rU; mU; rC; mC; rA; mC; rA; mU; rU; mC$

Data in table 6 shows that a compound with an inverted deoxy abasicmoiety on both sense and antisense strand (1) was more active then bluntended compound (4) or compound with inverted abasic on one of thestrands (2 or 3).

TABLE 7 Residual target Compound Concen- mRNA Sense 5->3 Sense AntisenseNo. name tration % Antisense 5->3 Modifications Modifications 1 CASP2_4_50 18 rG; mC; rC; mA; rG; mA; rA; mU; 2, 4, 6, 8, 10, 12, 2, 4, 6, 8,11, 13, S1001 20 22 rG; mU; rG; mG; rA; mA; rC; mU; rC; 14, 16, 18-2′-15, 17, 19-2′-  5 43 mC; rU; zidB$ OMe-3′- OMe-3′-Pi rA; mG; rG; mA; rG;mU; rU; mC; Pi; 20-cap- rC; rA; mC; rA; mU; rU; mC; rU; mG; idAb rG; mC$2, 4, 6, 8, 10, 12, 2, 4, 6, 8, 11, 13, 2 CASP2_4_ 50 28 rG; mC; rC; mA;rG; mA; rA; mU; 14, 16, 18-2′- 15, 17, 19-2′- S1002 20 35 rG; mU; rG;mG; rA; mA; rC; mU; rC; OMe-3′-Pi OMe-3′-  5 50 mC; rU$ Pi; 20-cap- rA;mG; rG; mA; rG; mU; rU; mC; idAb rC; rA; mC; rA; mU; rU; mC; rU; mG; rG;mC; zidB$ 3 CASP2_4_ 50 16 rG; mC; rC; mA; rG; mA; rA; mU; 2, 4, 6, 8,10, 12, 2, 4, 6, 8, 11, 13, S1000 20 21 rG; mU; rG; mG; rA; mA; rC; mU;rC; 14, 16, 18-2′- 15, 17, 19-2′-  5 44 mC; rU; zidB$ OMe-3′-Pi; 20-OMe-3′-Pi; 20- rA; mG; rG; mA; rG; mU; rU; mC; cap-idAb cap-idAb rC; rA;mC; rA; mU; rU; mC; rU; mG; rG; mC; zidB$ 4 CASP2_4_ 50 11 zidB; rG; rC;rC; rA; rG; rA; rA; rU; 0, 20-cap-idAb 2, 4, 6, 8, 11, 13, S1003 20 18rG; rU; rG; rG; rA; rA; rC; rU; rC; rC; 15, 17, 19-2′-  5 26 rU; zidB$OMe-3′- rA; mG; rG; mA; rG; mU; rU; mC; Pi; 20-cap- rC; rA; mC; rA; mU;rU; mC; rU; mG; idAb rG; mC; zidB$ 5 CASP2_4_ 50 23 zidB; rG; rC; rC;rA; rG; rA; rA; rU; 0, 20-cap-idAb 2, 4, 6, 8, 11, 13, S1004 20 36 rG;rU; rG; rG; rA; rA; rC; rU; rC; rC; 15, 17, 19-2′-  5 32 rU; zidB$OMe-3′-Pi rA; mG; rG; mA; rG; mU; rU; mC; rC; rA; mC; rA; mU; rU; mC;rU; mG; rG; mC$

Data in Table 7 shows that double stranded RNA compounds having aterminal cap (inverted deoxyabsic mioiety) at both the 3′ terminus ofthe antisense strand and 3′ terminus of the sense strand exhibitenhanced activity when compared to a dsRNA compound having a 3′ terminalcap covalently attached to the 3′ terminus of the antisense strand(compare compounds 2 and 3 and compounds 4 and 5).

TABLE 8 Residual target Compound concen- mRNA Sense 5->3 Sense AntisenseNo name tration % Antisense 5->3 Modifications Modifications 1 Myd88_11_  20 nM  7 rG; rA; rA; rU; rG; rU; rG; rA; 15, 16, 17, 18- 1, 4, 5, 6,9, 12, S1262   5 nM  7 rC; rU; rU; rC; rC; rA; rG2p; rA 2′-5′-bridge 13,15, 17, 18,   1 nM  20 2p; rC2p; rC2p; rA$ 19-2′-OMe-  0.5 nM  16 mU;rG; rG; mU; mC; mU; rG; 3′-Pi; 20-  0.1 nM  45 rG; mA; rA; rG; mU; mC;rA; C3; C3 mC; rA; mU; mU; mC; zc3p; zc3p$ 2 Myd88_11_   20 nM  11 rG;rA; rA; rU; rG; rU; rG; rA; 15, 16, 17, 18, 1, 4, 5, 6, 9, 12, S1266   5nM  11 rC; rU; rU; rC; rC; rA; rG2p; rA 19-2′-5′- 13, 15, 17, 18,   1 nM 9 2p; rC2p; rC2p; rA2p bridge; Phosphate 19-2′-OMe-  0.5 nM  10 mU; rG;rG; mU; mC; mU; rG; 3′-Pi; 20-  0.1 nM 126 rG; mA; rA; rG; mU; mC; rA;C3; C3 mC; rA; mU; mU; mC; zc3p; zc3p$ 3 Myd88_11_   20 nM  5 zc3p; rG;rA; rA; rU; rG; rU; rG; 15, 16, 17, 18, 1, 4, 5, 6, 9, 12, S1270   5 nM 6 rA; rC; rU; rU; rC; rC; rA; rG2p; 19-2′-5′- 13, 15, 17, 18,   1 nM 10 rA2p; rC2p; rC2p; rA2p bridge; 0-cap- 19-2′-OMe-  0.5 nM  31 mU; rG;rG; mU; mC; mU; rG; C3; Phosphate 3′-Pi; 20-  0.1 nM  47 rG; mA; rA; rG;mU; mC; rA; C3; C3 mC; rA; mU; mU; mC; zc3p; zc3p$ 4 Myd88_11_   20 nM 6 zc6Np; rG; rA; rA; rU; rG; rU; rG; 15, 16, 17, 18, 1, 4, 5, 6, 9, 12,S1274   5 nM  8 rA; rC; rU; rU; rC; rC; rA; rG2p; 19-2′-5′- 13, 15, 17,18,   1 nM  15 rA2p; rC2p; rC2p; rA2p bridge; 0-cap- 19-2′-OMe-  0.5 nM 22 mU; rG; rG; mU; mC; mU; rG; AmC6; 3′-Pi; 20-  0.1 nM  63 rG; mA; rA;rG; mU; mC; rA; Phosphate C3; C3 mC; rA; mU; mU; mC; zc3p; zc3p$ 5Myd88_11_   20 nM  11 rG; rA; rA; rU; rG; rU; rG; rA; — 1, 4, 5, 6, 9,12, S1276   5 nM  20 rC; rU; rU; rC; rC; rA; rG; rA; rC; 13, 15, 17, 18, 0.5 nM  57 rC; rA$ 19-2′-OMe- mU; rG; rG; mU; mC; mU; rG; 3′-Pi; 20-rG; mA; rA; rG; mU; mC; rA; C3; C3 mC; rA; mU; mU; mC; zc3p; zc3p$ 6Myd88_11_   20 nM  12 zidB; rG; rA; rA; rU; rG; rU; rG; 15, 16, 17, 18-1, 4, 5, 6, 9, 12, S1159   5 nM  9 rA; rC; rU; rU; rC; rC; rA; rG2p;2′-5′-bridge; 0- 13, 15, 17, 18,   1 nM rA2p; rC2p; rC2p; rA$ cap-idAb19-2′-OMe-  0.5 nM  24 mU; rG; rG; mU; mC; mU; rG; 3′-Pi; 20-  0.1 nM 57 rG; mA; rA; rG; mU; mC; rA; C3; C3 mC; rA; mU; mU; mC; zc3p; zc3p$ 7Myd88_11_   20 nM  6 zc3p; rG; rA; rA; dT; rG; dT; rG; 4, 6, 9, 10, 11,12, 1, 4, 5, 6, 9, 12, S1224   5 nM  13 rA; dC; dT; dT; dC; dC; rA; rG;13, 17, 18- 13, 15, 17, 18,   1 nM  33 rA; dC; dC; rA$ DNA-3′-Pi; 0-19-2′-OMe-  0.5 nM  37 mU; rG; rG; mU; mC; mU; rG; cap-C3 3′-Pi; 20- 0.1 nM  88 rG; mA; rA; rG; mU; mC; rA; C3; C3 mC; rA; mU; mU; mC; zc3p;zc3p$ 8 RAC1_2_   20 nM  39 rG; mA; rG; mU; rC; mC; rU; 2, 4, 6, 8, 10,12, 1, 3, 5, 7, 9, 11, S1324   5 nM  21 mG; rC; mA; rU; mC; rA; mU; 14,16, 18-2′- 13, 15, 17, 19- 1.25 nM  9 rU; mU; rG; mA; rA; zc3p; OMe-3′-2′-OMe-3′- 0.31 nM  27 zc3p$ Pi; 20-C3; C3 Pi; 20- mU; rU; mC; rA; mA;rA; mU; C3; C3; rG; mA; rU; mG; rC; mA; rG; Phosphate mG; rA; mC; rU;mC; zc3p; zc3p 9 RAC1_2_   20 nM  68 rG; mA; rG; mU; rC; mC; rU; 2, 4,6, 8, 10, 12, 1, 3, 5, 7, 9, 11, S1323   5 nM  26 mG; rC; mA; rU; mC;rA; mU; 14, 16, 18-2′- 13, 15, 17, 19- 1.25 nM  10 rU; mU; rG; mA; rA$OMe-3′-Pi 2′-OMe-3′- 0.31 nM  6 mU; rU; mC; rA; mA; rA; mU; Pi; 20- rG;mA; rU; mG; rC; mA; rG; C3; C3; mG; rA; mC; rU; mC; zc3p; Phosphate zc3p

Data in Table 8 shows that double stranded RNA compounds comprising a 3′terminal C3Pi-C3OH overhang covalently attached to the 3′ terminus ofthe antisense strand (guide strand) (Z=two C3 moieties) show excellentactivity (greater than 80% knock down at 20 μM) irrespective of themodifications on the complementary sense strand (see compounds 1-7).

Compounds 1-7 utilize sequences set forth in SEQ ID NOS:3 and 4 andcomprise a common antisense strand (SEQ ID NO:4) that includes two 3′terminal C3 moieties (C3Pi-C3OH) covalently attached to the 3′ terminalnucleotide and different sense strands which include variousmodifications disclosed in the present application. Compound 1 sensestrand includes unmodified ribonucleotides in positions 1-14 and 19 and2′5′ ribonucleotides in positions 15-18. Compound 2 sense strandincludes unmodified ribonucleotides in positions 1-14 and 2′5′ribonucleotides in positions 15-19 and includes a terminal phosphate(P(O)3). Compound 3 sense strand includes unmodified ribonucleotides inpositions 1-14 and 2′5′ ribonucleotides in positions 15-19 and includesa 3′ terminal C3Pi. Compound 4 sense strand includes unmodifiedribonucleotides in positions 1-14 and 2′5′ ribonucleotides in positions15-19 and includes a 3′ terminal amino C6 moiety covalently attached tothe 3′ terminal nucleotide. Compound 5 sense strand includes unmodifiedribonucleotides in positions 1-19 (each N′ is unmodified). Compound 6sense strand includes unmodified ribonucleotides in positions 1-14 and19 and 2′5′ ribonucleotides in positions 15-18 and includes a 3′terminal inverted abasic moiety covalently attached to the 3′ terminalnucleotide. Compound 7 sense strand includes unmodified ribonucleotidesin positions 1-3, 5, 7-8, 14-16 and 19 and deoxyribonucleotides inpositions 4, 6, 9, 10-13 and 17-18 and includes a 3′ terminal phosphate(Pi) and a C3OH moiety covalently attached at the 5′ terminus of theinverted abasic moiety covalently attached to the 3′ terminalnucleotide.

Compound 9 having two C3 moieties covalently attached to the 3′ terminusof the sense strand and of the antisense strand is more active than asimilar compound (8) having two C3 moieties covalently attached to the3′ terminus of the antisense strand.

Example 2 Delivery of Modified Nucleic Acid Molecules Targeting CASP2 tothe Retina

Assessment of Target Cell siRNA Delivery, Target Cell Gene KnockdownActivity and Specificity of Cleavage of Target Gene mRNAKnock Down of Target Gene is Measured in Target Tissue for ExampleFollowing Intravitreal Injection into the Rat Retina.

Background Different structural modifications were made in the siRNAtargeting the CASP2 gene, which are tested for exonuclease resistance.The aim of this study is to examine the in vivo distribution andactivity of the oligonucleotides including these modifications asdescribed below.

S1003 inv-dAb-GCCAGAAUGUGGAACUCCU-inv-dAb AGGAGUUCCACAUUCUGGC-inv-dAbS800 GCCAGAAUGUGGAACUCCU AGGAGUUCCACAUUCUGGC-C3Pi-C3OH

Description of the test material (S1003): RNA duplex with the followingstructure: Sense strand non-modified 19 mer with inverted-Abasic as 5′and 3′-cap. Anti-Sense strand 19-mer with 2′O-Me at positions 2, 4, 6,8, 11, 13, 15, 17 & 19 and with inverted Abasic as 3′-cap, Annealed.Quantity supplied: 336 μg Storage Conditions: −80° C.

Description of the test material (S800): RNA duplex with the followingstructure: Sense strand 19-mer with L-DNA at position 18. Anti-Sensestrand 19-mer with 2′OMe at positions 2, 4, 6, 8, 11, 13, 15, 17 & 19and two (CH2)3 propanediol at 3′ end (C3Pi-C3OH). Quantity supplied: 840μg. Storage Conditions: −80° C.

CNL: RNA duplex having same modifications as 5800 without a C3Pi-C3OHmoiety attached to the 3′ terminus.

Animals: Age: 6-8 week old male rats. 180-220 gr

Group Size: n=4/10; Total number of animals: 112

Animal Husbandry: Diet: Animals were provided an ad libitum commercialrodent diet (Harlan Teklad diet for rodents), and free access todrinking water.

Environment:

-   -   (i) Acclimatization of at least 5 days.    -   (ii) All the animals were confined in a limited access facility        with environmentally controlled housing conditions throughout        the entire study period, and maintained in accordance with HBI        approved standard operating procedures (SOPs). Automatically        controlled environmental conditions are set to maintain        temperature at 20-24° C. with a relative humidity (RH) of        30-70%, a 12-hr light/12-hr dark cycle and 15-30 air changes/hr        in the study room. Temperature, RH and the light cycle were        monitored by the control computer.

EXPERIMENTAL DESIGN is provided in Table 2-1

Time Group Delivery Route SiRNA Dose regime point Group No. Unilateral(LE) Type μg/10 μl/eye (days) Size I IVT S1003 20 μg/10 μl 1 4 II IVTS1003 20 μg/10 μl 3 4 III IVT S1003 20 μg/10 μl 7 4 IV IVT S800 20 μg/10μl 1 10 V IVT S800 20 μg/10 μl 3 10 VI IVT S800 20 μg/10 μl 7 10 VII IVTCNL 20 μg/10 μl 1 10 VIII IVT CNL 20 μg/10 μl 3 10 IX IVT CNL 20 μg/10μl 7 10 X IVT Vehicle 10 μl 1 10 XI IVT Vehicle 10 μl 3 10 XII IVTVehicle 10 μl 7 10 XIII Intact — N/A — N/A N/A 10

Study design: All animals from experimental groups I-XII were injectedIVT unilaterally into the Left Eye (LE)) at dose of 20 ng of testarticle or control (CNL) in 10 μl PBS vehicle or 10 μl vehicle only.Experimental group XIII will be used as intact control. The terminationstep will be accomplished according to the study design (1 day, 3 daysand 7 days after IVT treatment).

Anesthesia: Animals were anesthetized with an Isoflurane special circuitsystem (Stoelting, USA). Pupils will be dilated with Mydramid (0.5%tropicamide) eye drops. For additional topical anesthesia, will be usedLocalin (OXYBUPROCAINE HYDROCHLORIDE 0.4%). The Lacromycin (GentamycinSulfate (Equivalent to 0.3% Gentamycin base) ophthalmic solution toprevent/decrease post surgery inflammatory process.

Intravitreal injection was performed under a dissecting microscope. A30/33-gauge needle was used to make a punch incision 1 mm posterior tothe temporal limbus, and a syringe needle (30/33-gauge Insulin syringe0.3 ml, PIC 0.8 mm, Italy) was inserted through the incision, 1.5 mmdeep, as observed through the dilated pupil.

Scheduled euthanasia All animals were deeply anesthetized (Equithesine 4ml/kg I.P) and euthanized (decapitated) according to the study design(Table, Termination).

Tissue Collection: Both eyes from all animals were enucleated and storedon ice. The eyes will be dissected using a microscope, and grosspathologies will be graded according to sample grading scale (seeappendix 5 for “Eye Pathology Score”). The cornea will be puncturedusing a 27/30 G needle, to remove aqueous humor from the anteriorchamber. Using a microsurgical blade, a cut will be made along thelimbus, and the cornea and the lens removed. The remaining eyecup willbe opened by a sagittal cut through the sclera. The retina will beextracted from the eyecup, rinsed in PBS and separated. Using fine-tipforceps the retina will be collected into the appropriate test tube,frozen in liquid nitrogen, and transferred to the Molecular Biology Unitfor extraction of total RNA.

Evaluation

Knockdown activity of the siRNA targeting CASP2 in the rat retina wasdetermined by CASP2 mRNA expression level quantification using the qPCRmethod. CASP2_(—)4 siRNA cleavage site on the target gene will beverified by RACE and siRNA quantitation in the retina was performed byS&L qPCR (stem and loop qPCR).

Samples RNA Isolation: RNA were processed from retina samples accordingto standard procedures for total RNA isolation with EZRNA, by doubleextraction. CASP2_(—)4 siRNA quantification by qPCR: The delivery of theCASP2_(—)4 siRNA in the retina was measured by qPCR siRNAquantification. qPCR was performed according to standard methods usingSYBR Green method on Applied Biosystem 7300 PCR System. CASP2_(—)4 siRNAdirected cleavage of CASP2 mRNA in rat retina was determined by thedetection of the cleavage product using the RACE (Rapid Amplification ofcDNA Ends) method in the respective experimental groups. If evidence ofthe expected cleavage product is shown, the siRNA cleavage site on thetarget gene will be verified by sequence analysis and optionally thecleavage product will be quantified using qPCR. CASP2 mRNAquantification by qPCR: after cDNA is prepared CASP2 knock down will beverified by CASP2 mRNA quantification by qPCR. qPCR will be performedaccording to standard methods standard methods using SYBR Green methodon Applied Biosystem 7300 PCR System.

Preliminary Results

Preliminary results indicate that the 5800 is taken up more efficientlyinto the retinal cells than 51003. Results are shown in Table 2-2, below

TABLE 2-2 eye Structure Days N Mean Std Left CASP2_4_S1003 1 4 3.54 5.29CASP2_4_S1003 3 4 0.44 0.67 CASP2_4_S1003 7 4 0.27 0.17 CASP2_4_S800 110 26.16 17.65 CASP2_4_S800 3 10 1.52 1.93 CASP2_4_S800 7 10 0.81 0.74CASP2_4 CNL 1 10 2.53 2.31 CASP2_4 CNL 3 10 1.06 0.76 CASP2_4 CNL 7 90.10 0.08 Right CASP2_4_S1003 1 4 0.01 0.00 CASP2_4_S1003 3 3 0.01 0.00CASP2_4_S1003 7 4 0.02 0.02 CASP2_4_S800 1 10 0.14 0.26 CASP2_4_S800 310 0.18 0.41 CASP2_4_S800 7 10 0.10 0.09 CASP2_4 CNL 1 10 0.05 0.07CASP2_4 CNL 3 10 0.01 0.01 CASP2_4 CNL 7 9 0.01 0.01

Example 3 Delivery of Modified Nucleic Acid Molecules Targeting MYD88 tothe Retina

Assessment of Target Cell siRNA Delivery The objectives of the studyare:

1.1 To determine delivery of MYD88_(—)11 siRNA of different structuresand modifications to the rat retina 4 hours, one day and three daysfollowing unilateral Intravitreal (IVT) injections.1.2 To determine knockdown activity of the MYD88_(—)11 siRNA ofdifferent structures targeting MYD88 by means of qPCR of MYD88 mRNAafter IVT injection in the rat eyes 4 hours, one day and three daysafter IVT injections.

Background Different structural modifications were made in the siRNAtargeting the CASP2 gene, which are tested for exonuclease resistance.The aim of this study is to examine the in vivo distribution of doublestranded RNA oligonucleotides with modifications as disclosed herein,specifically the modifications described below.

MYD88_11 S505 5′GAAUGUGACUUCCAGAC c A 5′UGGUCUGGAAGUCACAUUCMYD88_11 S1159 5′idAb-GAAUGUGACUUCCAGACCA5′UGGUCUGGAAGUCACAUUC-C3Pi-C3OH MYD88_11 S12705′OHC3-GAAUGUGACUUCCAGACCA-Pi 5′UGGUCUGGAAGUCACAUUC-C3Pi-C3OH

Description of the test material S505: RNA duplex with the followingstructure: Sense strand Non-modified 19 mer with one L-DNA moiety inposition 18 (lower case bold).

Anti-Sense strand 19-mer with 2′O-Me at positions 2, 4, 6, 8, 11, 13,15, 17 & 19 Annealed. Storage Conditions: −80° C.

Description of the test material S1159: RNA duplex with the followingstructure: Sense strand 19-mer with inverted-Abasic as 5′ cap and 2′-5′bridged RNA at positions 15-18 The antisense strand is a 19-mer with2′O-Me at positions 1, 4-6, 9, 12-13, 15, 17-19 and two constitutiveunites of 1,3-Propanediol bond by phosphodiester bond at the 3′ end.).Annealed. Storage Conditions: −80° C.

Description of the test material S1270: RNA duplex with the followingstructure: Sense strand 19-mer with 2′5′ nucleotides in positions 15-19and a terminal phosphate (Pi). Anti-Sense strand 19-mer with 2′OMe atpositions 1, 4, 5, 6, 9, 12, 13, 15, 17-19 and two (CH2)3 propanediol at3′ end (C3Pi-C3OH). Annealed. Storage Conditions: −80° C.

Animals: Age: 8-10 week old male rats. 180-220 gr

Group Size: n=4/8; Total number of animals: 104

Animal Husbandry: Diet: Animals were provided an ad libitum commercialrodent diet (Harlan Teklad diet for rodents), and free access todrinking water.

Environment:

-   -   (i) Acclimatization of at least 5 days.    -   (ii) All the animals were confined in a limited access facility        with environmentally controlled housing conditions throughout        the entire study period, and maintained in accordance with HBI        approved standard operating procedures (SOPs). Automatically        controlled environmental conditions are set to maintain        temperature at 20-24° C. with a relative humidity (RH) of        30-70%, a 12-hr light/12-hr dark cycle and 15-30 air changes/hr        in the study room. Temperature, RH and the light cycle were        monitored by the control computer.

Study design: All animals from experimental groups 1-12 were injectedIVT unilaterally into the Left Eye (LE)) at dose of 20 μg of testarticle or 10 μl PBS vehicle only. Experimental group 13 was used asintact control. The termination step was accomplished according to thestudy design (4 hours, 1 day, and 7 days after IVT treatment).

EXPERIMENTAL DESIGN is provided in Table 3-1

TABLE 3-1 Group Delivery Route SiRNA Dose regime Time point Group No.Unilateral (LE) Type μg/eye (hours) Size 1 IVT MYD88_11_S505 20 μg/10 μl4 8 2 IVT MYD88_11_S505 20 μg/10 μl 24 8 3 IVT MYD88_11_S505 20 μg/10 μl168 8 4 IVT MYD88_11_S1159 20 μg/10 μl 4 8 5 IVT MYD88_11_S1159 20 μg/10μl 24 8 6 IVT MYD88_11_S1159 20 μg/10 μl 168 8 7 IVT MYD88_11_S1270 20μg/10 μl 4 8 8 IVT MYD88_11_S1270 20 μg/10 μl 24 8 9 IVT MYD88_11_S127020 μg/10 μl 168 8 10 IVT PBS 10 μl 4 8 11 IVT PBS 10 μl 24 8 12 IVT PBS10 μl 168 8 13 Intact — N/A — N/A N/A 8

Study design: All animals from experimental groups 1-12 were injectedIVT unilaterally into the Left Eye (LE)) at dose of 20 μg of testarticle or 10 μl PBS vehicle only. Experimental group 13 was used asintact control. The termination step was accomplished according to thestudy design (4 hours, 1 day, and 7 days after IVT treatment).

Anesthesia: Animals were anesthetized with an Isoflurane special circuitsystem (Stoelting, USA) working setup: 3-4.5% Isoflurane in 02 at600-800 ml/min 02 flow rate. Pupils were dilated with Mydramid (0.5%tropicamide) eye drops. For additional topical anesthesia, will be usedLocalin (OXYBUPROCAINE HYDROCHLORIDE 0.4%). The Lacromycin (GentamycinSulfate (Equivalent to 0.3% Gentamycin base) ophthalmic solution toprevent/decrease post surgery inflammatory process.

Intravitreal injection was performed under a dissecting microscope. A30/33-gauge needle was used to make a punch incision 1 mm posterior tothe temporal limbus, and a syringe needle (30/33-gauge Insulin syringe0.3 ml, PIC 0.8 mm, Italy) was inserted through the incision, 1.5 mmdeep, as observed through the dilated pupil.

Scheduled euthanasia All animals were deeply anesthetized (Equithesine 4ml/kg I.P) and euthanized (decapitated) according to the study design(Table, Termination).

Tissue Collection: Both eyes from all animals were enucleated and storedon ice. The eyes will be dissected using a microscope, and grosspathologies will be graded according to sample grading scale (seeappendix 5 for “Eye Pathology Score”). The cornea will be puncturedusing a 27/30 G needle, to remove aqueous humor from the anteriorchamber. Using a microsurgical blade, a cut will be made along thelimbus, and the cornea and the lens removed. The remaining eyecup willbe opened by a sagittal cut through the sclera. The retina will beextracted from the eyecup, rinsed in PBS and separated. Using fine-tipforceps the retina will be collected into the appropriate test tube,frozen in liquid nitrogen, and total RNA was extracted.

Evaluation

siRNA quantitation in the retina will be performed by Stem & Loop qPCRand Knockdown activity of the siRNA targeting MYD88 in the rat retinawill be determined by MYD88 mRNA expression level quantification usingqPCR.

RNA Isolation: RNA was processed from retina samples according tostandard procedures using the EZ RNA kit.

MYDD88 siRNA quantification by qPCR: The delivery of the MYDD88 siRNA inthe retina was measured by qPCR siRNA quantification (S&L). qPCR wasperformed according to standard procedures using the SYBR Green methodon Applied Biosystem 7300 PCR System.

MYDD88 mRNA quantification by qPCR: cDNA was prepared using standardprocedures and MYDD88 knock down will be verified by MYD88 mRNAquantification by qPCR. qPCR will be performed using SYBR Green methodon Applied Biosystem 7300 PCR System.

PRELIMINARY RESULTS are provided in Table 3-2, hereinbelow.

Delivery of the C3C3 modified compounds (S1159 and S1270) to retinalganglion cells was significantly higher than delivery of the compoundhaving blunt ends (S505) (see data provided in the “Median” column.Values are given as femtomolar).

TABLE 3-2 retina struct termination Delivery N Mean S.D. Median p.valueLeft MYD88_11_S1159 4 IVT 8 174 121 148 <.0001 MYD88_11_S1270 8 442 219466 MYD88_11_S505 8 52 38 38 MYD88_11_S1159 24 6 9 19 2 0.2928MYD88_11_S1270 5 37 74 5 MYD88_11_S505 8 2 3 0 MYD88_11_S1159 168 6 2 11 0.0034 MYD88_11_S1270 5 3 2 2 MYD88_1_S505 6 0 0 0 RightMYD88_11_S1159 4 IVT 4 2 1 1 0.0015 MYD88_11_S1270 0 * MYD88_11_S505 7 00 0 MYD88_11_S1159 24 4 14 23 2 0.3023 MYD88_11_S1270 5 6 10 2MYD88_11_S505 6 0 0 0 MYD88_11_S1159 168 7 6 10 1 0.2973 MYD88_11_S12704 3 2 2 MYD88_1_S505 7 0 0 0

Example 4 Model Systems of Acute Renal Failure (ARF)

ARF is a clinical syndrome characterized by rapid deterioration of renalfunction that occurs within days. Without being bound by theory theacute kidney injury may be the result of renal ischemia-reperfusioninjury such as renal ischemia-reperfusion injury in patients undergoingmajor surgery such as major cardiac surgery. The principal feature ofARF is an abrupt decline in glomerular filtration rate (GFR), resultingin the retention of nitrogenous wastes (urea, creatinine). Recentstudies, support that apoptosis in renal tissues is prominent in mosthuman cases of ARF. The principal site of apoptotic cell death is thedistal nephron. During the initial phase of ischemic injury, loss ofintegrity of the actin cytoskeleton leads to flattening of theepithelium, with loss of the brush border, loss of focal cell contacts,and subsequent disengagement of the cell from the underlying substratum.

Testing an active siRNA compound was performed using an animal model forischemia-reperfusion-induced ARF, as indicated in PCT patent applicationpublication No. WO/2009/044392.

An existing siRNA can be advantageously modified and future siRNA can bedesigned and produced to provide active nucleic acid molecules. In anon-limiting example siRNA compounds which utilize the oligonucleotidepairs set forth in Tables B (B1-B74), Tables C (C1-C4) and Tables D(D1-D34) of PCT Patent Publication No. WO/2009/044392, in particularsiRNAs directed to specific proapoptotic genes, in particular to genesTP53BP2, LRDD, CYBA, ATF3, CASP2, HRK, CIQBP, BNIP3, MAPK8, MAPK14,RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF,and SPP1) and further include and least one 3′ overhang according to thepresent invention are tested in the above model system and found to beprotective against ischemia reperfusion.

Example 5 Model Systems of Pressure Sores or Pressure Ulcers

Pressure sores or pressure ulcers including diabetic ulcers, are areasof damaged skin and tissue that develop when sustained pressure (usuallyfrom a bed or wheelchair) cuts off circulation to vulnerable parts ofthe body, especially the skin on the buttocks, hips and heels. The lackof adequate blood flow leads to ischemic necrosis and ulceration of theaffected tissue. Pressure sores occur most often in patients withdiminished or absent sensation or who are debilitated, emaciated,paralyzed, or long bedridden. Tissues over the sacrum, ischia, greatertrochanters, external malleoli, and heels are especially susceptible;other sites may be involved depending on the patient's situation.

Testing the active inhibitors of the invention (such as siRNA compounds)for treating pressure sore, ulcers and similar wounds is performed inthe mouse model as described in Reid et al., J Surg. Res. 116:172-180,2004.

An additional rabbit model is described by Mustoe et al, JCI, 1991.87(2):694-703; Ahn and Mustoe, Ann Pl Surg, 1991. 24(1):17-23, and isused for testing the siRNA compounds designed and synthesized asdisclosed herein. An existing siRNA can be advantageously modified andfuture siRNA can be designed and produced to provide active nucleic acidmolecules. In some embodiments siRNA compounds which utilize theoligonucleotide pairs set forth in Tables B (B1-B74), Tables C (C1-C4)and Tables D (D1-D34) of PCT patent application publication No.WO/2009/044392, and specifically compounds directed to genes CIQBP,RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, or TYROBP andfurther include and least one 3′ non-nucleotide overhang according tothe present invention are tested in animal models where it is shown thatthese siRNA compounds treat and prevent pressure sores and ulcers.

Example 6 Model Systems of Chronic Obstructive Pulmonary Disease (COPD)

Chronic obstructive pulmonary disease (COPD) is characterized mainly byemphysema, which is permanent destruction of peripheral air spaces,distal to terminal bronchioles. Emphysema is also characterized byaccumulation of inflammatory cells such as macrophages and neutrophilsin bronchioles and alveolar structures. Emphysema and chronic bronchitismay occur as part of COPD or independently.

Testing the active inhibitors of the invention (such as siRNA) fortreating COPD/emphysema/chronic bronchitis is performed in animal modelssuch as those disclosed as follows:

Starcher and Williams, 1989. Lab. Animals, 23:234-240; Peng, et al.,2004.; Am J Respir Crit Care Med, 169:1245-1251; Jeyaseelan et al.,2004. Infect. Immunol, 72: 7247-56. Additional models are described inPCT patent publication WO 2006/023544 assigned to the assignee of thepresent application, which is hereby incorporated by reference into thisapplication.

An existing siRNA can be advantageously modified and future siRNA can bedesigned and produced to provide active nucleic acid molecules. In someembodiments siRNA compounds which utilize the oligonucleotide pairs setforth in Tables B (B1-B74), Tables C (C1-C4) and Tables D (D1-D34) ofPCT patent application publication No. WO/2009/044392, and in particularto siRNA to genes CIQBP, BNIP3, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4,BMP2, CX43, TYROBP, CTGF, and DUOX1 and further include and least one 3′overhang as disclosed herein are tested in these animal models, whichshow that these siRNA compounds may treat and/or prevent emphysema,chronic bronchitis and COPD.

Example 7 Model Systems of Spinal Cord Injury

Spinal cord injury, or myelopathy, is a disturbance of the spinal cordthat results in loss of sensation and/or mobility. The two common typesof spinal cord injury are due to trauma and disease. Traumatic injurycan be due to automobile accidents, falls, gunshot, diving accidentsinter alia, and diseases which can affect the spinal cord include polio,spina bifida, tumors and Friedreich's ataxia.

An existing siRNA can be advantageously modified and future siRNA can bedesigned and produced to provide active nucleic acid molecules. In someembodiments siRNA compounds which utilize the oligonucleotide pairs setforth in 7 Tables B (B1-B74), Tables C (C1-C4) and Tables D (D1-D34) ofPCT patent application publication No. WO/2009/044392, and in particularsiRNA directed to genes LRDD, CYBA, ATF3, CASP2, HRK, CIQBP, BNIP3,MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2,CX43, TYROBP, CTGF, and RHOA and further include and least one 3′overhang as disclosed herein are tested in this animal model, which showthat these siRNA compounds promote functional recovery following spinalcord injury and thus may be used to treat spinal cord injury.

Example 8 Model Systems of Glaucoma

Testing the active inhibitors of the invention (such as siRNA) fortreating or preventing glaucoma is done in the animal model for exampleas described by Pease et al., J. Glaucoma, 2006, 15(6):512-9 (Manometriccalibration and comparison of TonoLab and TonoPen tonometers in ratswith experimental glaucoma and in normal mice).

An existing siRNA can be advantageously modified and future siRNA can bedesigned and produced to provide active nucleic acid molecules. In someembodiments siRNA compounds which utilize the oligonucleotide pairs setforth in Tables B (B1-B74), Tables C (C1-C4) and Tables D (D1-D34) ofPCT patent application publication No. WO/2009/044392, in particular togenes TP53BP2, LRDD, CYBA, ATF3, CASP2, HRK, BNIP3, MAPK8, MAPK14, RAC1,and RHOA and further include and least one 3′ non-nucleotide overhang asdisclosed herein are tested in this animal model which show that thesesiRNA compounds treat and/or prevent glaucoma.

Example 8A Model Systems of Ischemic Optic Neuropathy (ION)

An animal model for Ischemic optic neuropathy was established in adultsWistar rats using a protocol of optic nerve crush injury. Seven daysprior to the optic nerve crush, the retinal ganglion cells (RGC) areselectively labelled by application of the retrograde tracer FluoroGold(2%, Fluorochrome, Englewood, Colo.) to the superior colliculus. Thetracer is transported by retrograde transport along RGC axons resultingin complete and specific labeling of all RGCs within 1 week postinjection of the fluorescent tracer. The animals are subjected to theoptic nerve crush injury 7 days post retrograde tracing. The orbitaloptic nerve is exposed through a supraorbital approach and all axons inthe optic nerve are transected by crushing with forceps for 10 seconds,2 mm from the lamina cribrosa. A single dose of 20 μg/5 μl of PBS of ansiRNA compound according to the invention is microinjected into thevitreous body 2 mm anterior to the nerve head, using a glassmicropipette at the time of the optic nerve crush The survival of RGCsis determined 7 days following the optic nerve crush by countingFluoroGold-labelled RGCs on flat-mounted retinas. The experimentalanimals are perfused transcardially with 4% paraformaldehyde at 1 weekafter the optic nerve crash. Both retinas are dissected out, fixed foran additional 30 min and flat-mounted on a glass slide for ganglion celllayer quantification. The number of fluorescent RGCs is counted in 16distinct areas in each retina and the percent of survival of the RGCs isdetermined compared to samples obtained from rats which did not undergooptic nerve crush injury at all or samples obtained from rats which wereinjected with PBS, control siRNA or GFP siRNA along with the optic nervecrush injury. Microglia cells that may have incorporated FluoroGoldafter phagocytosis of dying RGCs were distinguished by theircharacteristic morphology and excluded from quantitative analyses.

Another model of optic nerve axotomy where the entire population of RGCsare axotomized by transecting the optic nerve close to the eye is usefulfor testing the compounds and compositions of the present invention.(Cheng L, et al. J. Neurosci. May 15, 2002 2002;22:3977-3986).

Example 9 Model Systems of Ischemia/Reperfusion Injury Following LungTransplantation in Rats

Testing the active inhibitors of the invention (such as siRNA) fortreating or preventing ischemia/reperfusion injury or hypoxic injuryfollowing lung transplantation is done in one or more of theexperimental animal models, for example as described by Mizobuchi etal., 2004. J. Heart Lung Transplant, 23:889-93; Huang, et al., 1995. J.Heart Lung Transplant. 14: S49; Matsumura, et al., 1995. Transplantation59: 1509-1517; Wilkes, et al., 1999. Transplantation 67:890-896; Naka,et al., 1996. Circulation Research, 79: 773-783.

An existing siRNA can be advantageously modified and future siRNA can bedesigned and produced to provide active nucleic acid molecules. In someembodiments siRNA compounds which utilize the oligonucleotide pairs setforth in Tables B (B1-B74), Tables C (C1-C4) and Tables D (D1-D34) ofPCT patent application publication No. WO/2009/044392, and in particularto TP53BP2, LRDD, CYBA, CASP2, BNIP3, RAC1, and DUOX1 and furtherinclude and least one 3′ non-nucleotide overhang as disclosed herein aretested in these animal models, which show that these siRNA compoundstreat and/or prevent ischemia-reperfusion injury following lungtransplantation and thus may be used in conjunction with transplantsurgery.

Example 10 Model Systems of Acute Respiratory Distress Syndrome

Testing the active inhibitors of the invention (such as siRNA) fortreating acute respiratory distress syndrome is done in the animal modelas described by Chen et al (J Biomed Sci. 2003; 10(6 Pt 1):588-92. siRNAcompounds according to Tables B (B1-B74), Tables C (C1-C4) and Tables D(D1-D34) of PCT patent application publication No. WO/2009/044392, inparticular to genes CYBA, HRK, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7,TRPM2, PARG, SPP1, and DUOX1 and further include and least one 3′overhang as disclosed herein are tested in this animal model which showsthat these siRNAs treat and/or prevent acute respiratory distresssyndrome and thus may be used to treat this condition.

Example 11 Animal Models of Osteoarthritis (OA)

Collagen induced arthritis (CIA): CIA in mice is described in Trenthamet al. (1977. J. Exp. Med. 146: 857-868). Adjuvant-induced arthritis(AA):AA is described in Kong et al., (1999. Nature, 402:304-308). Amenisectomy model is described in Han et al., (1999. Nagoya J Med Sci62(3-4):115-26).

The effect of different siRNA inhibitors, such as siRNA to SSP1, ondifferent parameters related to OA such as chondrocyte proliferation,terminal differentiation and development of arthritis, is evaluatedusing one or more of the above models, in addition to in vitro modelsknown in the art. siRNA compounds directed to specific proapoptoticgenes, in particular to SSP1, and further include and least one 3′overhang as disclosed herein are tested in these animal models whichshow that these siRNAs treat and/or prevent OA and thus may be used totreat this condition.

Example 12 Rat Model Systems for Transplantation-Associated Acute KidneyInjury

Warm Ischemia—

In test rats a left nephrectomy is performed, followed by autotransplantation that results in a warm kidney graft preservation periodof 45 minutes. Following auto transplantation, a right nephrectomy isperformed on the same animal. Chemically modified siRNA to a target isadministered intravenously via the femoral vein either before harvestingof the kidney graft (mimicking donor treatment) (“pre”), or after thekidney autotransplantation (mimicking recipient treatment), or bothbefore harvest and after transplantation (combined donor and recipienttreatment) (“pre-post”).

Cold Ischemia—

A left nephrectomy is performed on a donor animal, followed by a coldpreservation (on ice) of the harvested kidney for a period of 5 hours.At the end of this period, the recipient rat will undergo a bilateralnephrectomy, followed by transplantation of the cold-preserved kidneygraft. The total warm ischemia time (including surgical procedure) isabout 30 minutes. Chemically modified siRNA is administeredintravenously via the femoral vein, either to the donor animal prior tothe kidney harvest (“pre”), or to the recipient animal 15 minutes (“post15 min”) or 4 hours (post 4 hrs) post-transplantation.

To assess the efficacy of siRNA in improvement of post-transplantationrenal function, serum creatinine levels are measured on days 1, 2, and 7post-transplantation in both warm and cold ischemia models.

1. A double-stranded nucleic acid molecule comprising a sense strandsequence 5′ CAGAAGUCAUCUUGCUACA 3′ (SEQ ID NO. 9) and an antisensestrand sequence 5′ UGUAGCAAGAUGACUUCUG 3′ (SEQ ID NO. 10); or apharmaceutically acceptable salt of such molecule.
 2. Thedouble-stranded nucleic acid molecule of claim 1, wherein the antisensestrand comprises a non-nucleotide overhang covalently attached via aphosphodiester or a phosphorothioate linkage to the 3′ or to the 2′position of the sugar residue of the 3′ terminal nucleotide, wherein thenon-nucleotide overhang is selected from the group consisting of C3Pi,C3Ps, C3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi, C3Ps-C3Ps,C3Pi-C3Pi-C3OH, C3Ps-C3Ps-C3OH, C3Pi-C3Ps-C3OH, C3Ps-C3Pi-C3OH,C3Pi-C3Pi-C3Pi, C3Ps-C3Ps-C3Ps, C3Pi-C3Ps-C3Ps, C3Ps-C3Pi-C3Ps,C3Ps-C3Ps-C3Pi, C3Pi-C3Pi-C3Ps, C3Ps-C3Pi-C3Pi, C3Pi-C3Ps-C3Pi,C3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPi-C3Pi,rAbPs-C3OH, rAbPs-C3Pi, dAbPi-C3OH, dAbPi-C3Pi, dAbPs-C3OH anddAbPs-C3Pi; wherein each Pi is a phosphodiester linkage and each Ps is aphosphorothioate linkage; or a pharmaceutically acceptable salt of suchmolecule.
 3. The double-stranded nucleic acid molecule of claim 2 havingstructure (A1) (A1) 5′(N)x-Z3′     (antisense strand)3′Z′-(N′)y-z″5′(sense strand)

wherein each of N and N′ is an unmodified ribonucleotide, a modifiedribonucleotide, or an unconventional moiety; wherein each of (N)x and(N′)y is an oligonucleotide in which each consecutive N or N′ is joinedto the next N or N′ by a covalent bond; wherein Z comprises anon-nucleotide overhang covalently attached via a phosphate basedlinkage to the 3′ position or the 2′ position of the sugar residue ofthe 3′ terminal nucleotide in the antisense strand; wherein Z comprisesa non-nucleotide overhang selected from the group consisting of C3Pi,C3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps, C3Ps-C3OH, C3Ps-C3Pi, C3Ps-C3Ps,C3Pi-C3Pi-C3OH, C3Ps-C3Ps-C3OH, C3Pi-C3Ps-C3OH, C3Ps-C3Pi-C3OH,C3Pi-C3Pi-C3Pi, C3Ps-C3Ps-C3Ps, C3Pi-C3Ps-C3Ps, C3Ps-C3Pi-C3Ps,C3Ps-C3Ps-C3Pi, C3Pi-C3Pi-C3Ps, C3Ps-C3Pi-C3Pi, C3Pi-C3Ps-C3Pi,C3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPi-C3Pi,rAbPs-C3OH, rAbPs-C3Pi, dAbPi-C3OH, dAbPi-C3Pi, dAbPs-C3OH anddAbPs-C3Pi; wherein each Pi is a phosphodiester linkage and each Ps is aphosphorothioate linkage; wherein Z′ is present or absent; but ifpresent, Z′ is an inorganic phosphate or a non-nucleotide overhangselected form the group consisting of an abasic moiety, an invertedabasic moiety and an alkyl moiety or a derivative thereof or acombination thereof; covalently attached via a phosphate based linkageto the 3′ position or the 2′position of the sugar residue of the 3′terminal nucleotide in the sense strand; wherein z″ may be present orabsent, but if present is a capping moiety covalently attached at the 5′terminus of (N′)y; wherein each of x and y is independently an integerbetween 18 and 40; wherein the sequence of (N′)y has complementarity tothe sequence of (N)x; and wherein the sequence of (N)x hascomplementarity to a consecutive sequence in a target RNA; or apharmaceutically acceptable salt of such molecule.
 4. The molecule ofclaim 3, wherein x=y=19.
 5. The molecule of claim 3, wherein Z′ ispresent.
 6. The molecule of claim 3, wherein Z comprises anon-nucleotide overhang selected from the group consisting of C3Pi-C3OH,C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3Pi, C3Ps-C3OH and C3Ps-C3Ps.
 7. Themolecule of claim 6, wherein Z′ is a non-nucleotide overhang selectedfrom the group consisting of C3OH, C3Pi and C3Ps, wherein Pi is aphosphodiester linkage and Ps is a phosphorothioate linkage.
 8. Themolecule of claim 2, wherein the non-nucleotide overhang is covalentlyattached to the 3′ position of the sugar residue of the 3′ terminalnucleotide such that the 3′ terminal nucleotide has one of the followingstructures:

wherein B is a nucleotide base and R is H, OH or a 2′ sugarmodification.
 9. The molecule of claim 1, wherein the non-nucleotideoverhang is selected from the group consisting of C3Pi-C3OH, C3Pi-C3Pi,C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi and C3Ps-C3Ps.
 10. The molecule of claim1, wherein the non-nucleotide overhang is selected from the groupconsisting of —C3Pi-C3Pi-C3OH, —C3Ps-C3Ps-C3OH, —C3Pi-C3Ps-C3OH,—C3Ps-C3Pi-C3OH, —C3Pi-C3Pi-C3Pi, —C3Ps-C3Ps-C3Ps, —C3Pi-C3Ps-C3Ps,—C3Ps-C3Pi-C3Ps, —C3Ps-C3Ps-C3Pi, —C3Pi-C3Pi-C3Ps, —C3Ps-C3Pi-C3Pi and—C3Pi-C3Ps-C3Pi.
 11. The molecule of claim 1, wherein the non-nucleotideoverhang is selected from the group consisting of —C3Pi-rAb, —C3Pi-dAb,—C3Ps-rAb and —C3Ps-dAb.
 12. The molecule of claim 1, further comprisinga non-nucleotide overhang covalently attached to the 3′ terminalnucleotide in the sense strand, wherein the non-nucleotide overhangcovalently attached to the 3′ terminal nucleotide in the sense strand isselected from the group consisting of propanol, a C3 alkyl moiety linkedto a phosphodiester and a C3 alkyl moiety linked to a phosphorothioate.13. The molecule of claim 12, wherein the non-nucleotide overhangcovalently attached to the 3′ terminal nucleotide in the sense strand ispropanol; and wherein the 3′ terminal sugar residue has the followingstructure:

wherein B is a nucleotide base and R is H, OH or a 2′ sugarmodification.
 14. The molecule of claim 12, wherein the non-nucleotideoverhang covalently attached to the 3′ terminal nucleotide in the sensestrand is C3 alkyl moiety linked to a phosphodiester; and wherein the 3′terminal sugar residue has the following structure:

wherein B is a nucleotide base and R is H, OH or a 2′ sugarmodification.
 15. The molecule of claim 12, wherein the non-nucleotideoverhang covalently attached to the 3′ terminal nucleotide in theantisense strand is selected from the group consisting of C3Pi-C3OH,C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi and C3Ps-C3Ps; and whereineach Pi is a phosphodiester linkage and each Ps is a phosphorothioatelinkage.
 16. The molecule of claim 15, wherein the non-nucleotideoverhang covalently attached to the 3′ terminal nucleotide in theantisense strand is C3Pi-C3OH; and wherein the 3′ terminal sugar residuehas the following structure:

wherein B is a nucleotide base and R is H, OH or a 2′ sugarmodification.
 17. The molecule of claim 15, wherein the non-nucleotideoverhang covalently attached to the 3′ terminal nucleotide in theantisense strand is C3Pi-C3Pi; and wherein the 3′ terminal sugar residuehas the following structure:

wherein B is a nucleotide base and R is H, OH or a 2′ sugarmodification.
 18. (canceled)
 19. (canceled)
 20. The double strandednucleic molecule of claim 5, wherein Z′ is a non-nucleotide overhangselected from the group consisting of C3OH, C3Pi, C3Ps, C3Pi-C3OH,C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi, C3Ps-C3Ps, C3Pi-C3Pi-C3OH,C3Ps-C3Ps-C3OH, C3Pi-C3Ps-C3OH, C3Ps-C3Pi-C3OH, C3Pi-C3Pi-C3Pi,C3Ps-C3Ps-C3Ps, C3Pi-C3Ps-C3Ps, C3Ps-C3Pi-C3Ps, C3Ps-C3Ps-C3Pi,C3Pi-C3Pi-C3Ps, C3Ps-C3Pi-C3Pi, C3Pi-C3Ps-C3Pi, C3Pi-rAb, C3Pi-dAb,C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPi-C3Pi, rAbPs-C3OH, rAbPs-C3Pi,dAbPi-C3OH, dAbPi-C3Pi, dAbPs-C3OH and dAbPs-C3Pi.
 21. The doublestranded nucleic molecule of claim 5, wherein Z′ is an inorganicphosphate.
 22. A composition comprising the molecule of claim 1 or apharmaceutically acceptable salt of such molecule, and apharmaceutically acceptable carrier.
 23. (canceled)